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1.  An Instrumental and Software Platform for The Targeted Protein-Protein Interact-ome of Signaling Pathways from Drosophila to Human Cancer Cells 
Journal of Biomolecular Techniques : JBT  2010;21(3 Suppl):S34-S35.
RP-46
The insulin signaling pathway in eukaryotes is central to gene translation, cell growth and proliferation. Drosophila is a sophisticated model system for signal transduction and systems biology studies. We have dissected the MAPK and AKT branches of the insulin signaling pathway through immunprecipitations of 38 key nodal proteins and label-free quantitative shotgun mass spectrometry of the prey proteins. These bait-prey experiments produced a protein interact-ome showing many canonical interactions in addition to many novel interactions. Bait proteins such as the catalytic regulatory subunits of phosphoinositide-3-kinase (PI3K) and insulin receptor substrate (IRS) were also carried out in human cancer cell lines and compared to the fly data. This revealed several novel pathway members. For the drosophila insulin signaling pathway, tandem affinity purification (TAP) and shotgun LC/MS/MS using an EASY-nLC and Orbitrap XL-ETD were performed under basal, insulin and EGF stimulation including TAP controls. The protein-protein interaction network was assembled by label-free quantification of the bait-prey interactions using MS/MS based average TIC across peptides identifying each prey protein and spectral counting using in-house developed NakedQuant v1.1 software that contains a protein grouping algorithm, normalization function and network assembly algorithm. For human cells, multiple myeloma and small cell lung cancer cell lines were prepared by immunoprecipitating (IP) several insulin signaling bait proteins including PI3K, IRS and TSC from basal and kinase inhibitor drug treated states. The bait-prey interaction results between human and fly were compared with a focus on label-free quantitative differences of signal changes between stimulating conditions. Bait-prey protein lists were compared by reverse BLAST and in-house developed tools in order to find similar novel interacting proteins between fly and human cells. A comprehensive interactome has been assembled. The pathway revealed several proteins that are novel and critical components of the activation pathway in addition to many canonical interactions. The criteria for true novel protein binding are 1.) a significant change in signal upon stimulation or drug treatment and 2.) the prey protein must be present from IPs from both drosophila cells and human cells. This ensures that the interaction is conserved over evolutionary time. In addition, we have biochemical validation of novel interactions in cancer cells using both Western blots and shRNAi in fly and cancer cells.
PMCID: PMC2918029
2.  A Mass Spectrometry Platform to Quantitatively Profile Cancer Cell Metabolism from Cell Lines to Tissues 
Journal of Biomolecular Techniques : JBT  2010;21(3 Suppl):S39-S40.
RP-60
The metabolic requirements of cancer and proliferating cells are different from that of normal differential tissue (the Warburg effect) and may have diverse applications in the treatment of cancers and other neoplastic diseases. However, many of the molecular mechanisms that conspire to reorganize metabolism to support cell proliferation are unknown. To study the mechanisms of cancer cell metabolism, we have implemented a mass spectrometry based platform to robustly quantitatively profile endogenous metabolites from proliferating cell lines and tumor tissues to extensively study cancer cell metabolism. Cell lines are derived from several cancers including lung, multiple myeloma and prostate, as well as from a fast proliferating Drosophila cell line. In addition, endogenous and Xenograft tumor tissue from tumors such as prostate are profiled before and after drug treatments. We routinely target nearly 250 metabolites using multiple reaction monitoring (MRM) based analyses with an AB/Sciex 5500 QTRAP mass spectrometer coupled to a Shimadzu UFLC using normal phase Hydrophilic interaction chromatography (HILIC) at pH=9.0 with positive/negative switching within the same experimental 25 minute LC/MS/MS run. For a single experiment, our platform allows for unprecedented sensitivity, quantitation and coverage of metabolites that comprise of diverse metabolic pathways from as little as a single 6 cm tissue culture dish of cells or approximately 3 million cells from tissue samples. We find that 2.00mm id x 10cm Luna NH2 HILIC columns (Phenomenex) at 250uL/min perform well in both negative and positive ion mode and that the sampling rate of the instrument is sufficiently fast to effectively capture up to 300 metabolite targets within approximately a 20-25 minute gradient without the need for scheduled MRM runs resulting in a cycle time of approximately 2 seconds with 5ms dwell times. Peak areas of metabolites are integrated post run using MultiQuant 1.1 software (Applied Biosystems). Peak areas from triplicate runs are then clustered using hierarchical clustering and statistical analyses are applied in order to generate P values for metabolite changes over different cellular conditions. We have also carried out preliminary studies to probe flux in pathways by targeting a set of 13C labeled metabolites from experiments where 13C labeled glucose is added to cells. Using this platform, we have observed the metabolic effects of growth factor signaling by analyzing metabolites from serum-starved versus serum-fed cells derived from several cancers. We have also analyzed the metabolism of a Drosophila model cell line after stimulation with Insulin and EGF (Spitz) to examine if growth factor induced metabolic changes are evolutionarily conserved. Using metabolic inhibitors, such as Iodoacetic acid and KCN, we have also been able to characterize the consequences of inhibiting glycolysis and oxidative phosphorylation, respectively. Finally, we considered a dual-pan PI3K/mTOR catalytic site inhibitor and measured its effects on metabolism in a proliferating breast epithelial cell line. In addition, we have profiled cerebral spinal fluid (CSF) from gliobastoma (GBM) patients and noticed several metabolioc profiles that are unique to GBM patients with mutated genes.
PMCID: PMC2918204
3.  A functional genomic analysis of cell morphology using RNA interference 
Journal of Biology  2003;2(4):27.
Background
The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies.
Results
We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA.
Conclusions
Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.
doi:10.1186/1475-4924-2-27
PMCID: PMC333409  PMID: 14527345
4.  A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist. 
Molecular and Cellular Biology  1995;15(9):5123-5130.
We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.
PMCID: PMC230759  PMID: 7651429
5.  Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster. 
Molecular and Cellular Biology  1990;10(6):3232-3238.
The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.
Images
PMCID: PMC360688  PMID: 2111451
6.  Drosophila melanogaster homologs of the raf oncogene. 
Molecular and Cellular Biology  1987;7(6):2134-2140.
A murine v-raf probe, representing the kinase domain, was used to identify two unique loci in Drosophila melanogaster DNA. The most closely related to v-raf was mapped by in situ hybridization to position 2F5-6 (Draf-1) on the X chromosome, whereas the other raf-related gene (Draf-2) was found at position 43A2-5 on chromosome 2. The nucleotide and amino acid homologies of Draf-1 to the kinase domain of v-raf are 61 and 65%, respectively. The large amount of a 3.2-kilobase Draf-1 transcript detected in eggs as a maternal message decreases during embryonic development, and significant steady-state levels are observed throughout the remainder of morphogenesis. We speculate that the Draf-1 locus plays an important role in early embryogenesis.
Images
PMCID: PMC365335  PMID: 3037346

Results 1-6 (6)