GFP Used to Selectively Label Tumor Cells in vivo.
The ability to selectively target and label tumor cells and tissues in vivo has eluded researchers and clinicians for decades. Recent work from a team of researchers from the laboratories of MIT, UCSD, Okayama University Graduate School of Medicine (JAPAN), Okayama University Hospital, and two companies, AntiCancer, Inc. (San Diego, CA) and Oncolys BioPharma, Inc. (Tokyo, Japan) have developed a technique to label tumors with the Green Fluorescent Protein (GFP) by selectively activating transcription of GFP within the cells using a telomerase reverse transcriptase promoter (hTERT). Telomerase is known to be active in malignant tissues. In this way they were able to label and identify even single tumor cells. They developed a telomerase-dependent, replication-competent adenovirus (OBP-401) that expressed GFP only in the cancer cells in vivo. The technique was used to identify A549 human lung cancer cells (that were cloned to also express red fluorescent protein; RFP) and showed that the labeling patterns were co-localized in a nude mice model. They also tested this new system for identifying HCT-116 human coloretal tumor cells in the peritoneum of mice with success. The implications of this method of using marker genes to identify cancer in human patients, as an aid in sugical removal and especially for identification of small metastatic tumors is significant. For more information about these new techniques please see the references below or visit our website.
- Kishimoto H, Zhao M, Hayashi K, Urata Y, Tanaka N, Fujiwara T, Penman S, Hoffman RM (2009) "In vivo internal tumor illumination by telomerase-dependent adenoviral GFP for precise surgical navigation." PNAS 106(34): 14514-14517.
- Kishimoto H, Urata Y, Tanaka N, Fujiwara T, Hoffman R (2009) "Selective metastatic tumor labeling with green fluorescent protein and killing by systemic administration of telomerase-dependent adenoviruses." Molecular Cancer Therapeutics 8:3001-3008.
- Kishimoto H, Kojima T, WatanabeY, Kagawa S, Fujiwara T, Uno F, Teraishi F, Kyo S, Mizuguchi H, HashimotoY, Urata Y, Tanaka N, Fujiwara T (2006) "In vivo imaging of lymph node metastasis with telomerase specific
replication-selective adenovirus." Nature Med. 12:1213–1219.
Improved anti-Photobleaching Media for GFPs.
 The use of Green Fluorescent Proteins (GFPs) and other colored marker gene homologs in cell biology has become a powerful tool for monitoring the expression levels and distribution of genes in live cell systems. But significant photobleaching or wavelength shifts of the proteins can occur in some instances. Recent work from the Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry (Moscow, Russia) and Evrogen, JSC have defined several new cell culture media that show improved photostability for GFP analyses. These media are based on the common DMEM (Dulbecco's Modified Eagle Medium) in wide use in cell culture laboratories, with several components, namely either riboflavin (DMEM-Rf) or several vitamins (DMEM-V) removed. These components were found to be efficient electron acceptors, and since the GFPs are good electron donors, their presence in the media was found to enhance photobleaching, reduce photostability, and result in conversion of the proteins into a red fluorescent state. Among the vitamins that were found to be important in photobleaching were L-Inositol, D-Ca pantothenate, Choline chloride, Folic acid, Nicotinamide, Pyridoxal HCl, Thiamine HCl as well as Riboflavin. For many different GFPs, as well as GFP-conjugates, they observed up to a 9-fold improvement in photostability. For more information about these new media and systems please see the references below or visit our website.
- Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S, Lukyanov KA, (2009) "Cell culture medium affects GFP photostability: a solution." Nature Methods 6: 859-860.
- Cormack, BP, Valdivia RH, Falkow S "FACS-optimized mutants of the green
fluorescent protein (GFP)." Gene 173: 33-38 (1996).
- Gurskay, NG Fradkov AF, Pounkova NI, Staroverov DB, Bulina ME, Yanushevich YG, Labas YA, Lukyanov S, Lukyanov KA, (2003) "A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants." Biochem. J. 373: 403-408.
- Subach, OM, et al. (2008) "Conversion of red fluorescent protein into a bright blue probe." Chem Biol 15: 1116-1124.
- Patterson GH, Lippincott-Schwartz J (2002) "A photoactivatable GFP for selective photolabeling of proteins and cells." Science 297: 1873-1877.
- Inouye S, Tsuji FL (1994) "Evidence for redox forms of the Aequorea green fluorescent protein." FEBS Lett. 351(2):211-214.
Oxidative Burst in Neutrophils with DHR123.
Reduced or missing oxidative burst activity in leukocytes is an indication of hereditary diseases such as chronic granulomatous disease (CGD). Treatment of cells (e.g. heparinized whole blood) with the reduced dye DHR123, (dihydrorhodamine 123, M0545) has been found to be a sensitive assay for measuring oxidative activity in such cells, with allied induction using either the chemotactic peptide N-formyl-Met-Leu-Phe (fMLP), the protein kinase C ligand phorbol 12-myristate-13-acetate (PMA) or by bacterial challenge. DHR123 is practically non-fluorescent until oxidized intracellularly to the bright red fluorescent rhodamine 123 product. Staining with DHR123 has been used to discern Chronic Granulomatous Disease (CGD) as well as complete myeloperoxidase deficiency, both of which yield strongly reduced dihydrorhodamine 123 staining compared with normal tissues. In addition, DHR123 has been found to be superior to other substrates, such as 2,7-dichlorodihydrofluorescein diacetate or dihydrorhodamine 6G in detecting intracellular hydrogen peroxide production in tumor cells. Allied methods for detection of ROS activity include luminol amplified chemiluminescence or cytochrome c reduction by UV/vis spectrometry. For more information about these assays, please see the references listed below or visit our website:
- Freitas M, Porto G, Lima, JLFC, Fernandes E, (2009) "Optimization of experimental settings for the analysis of human neutrophils oxidative burst in vitro." Talanta 78(4-5): 1476-1483.
- Dobmeyer, T.S., Raffel, B. Dobmeyer, J.M., Findhammer, S., Klein, S.A., Kabelitz, D. Hoelzer, D., Helm, E.B. & Rossol.(1995) Decreased function of monocytes and granulocytes during HIV-1 infection correlates with CD4 cell counts. Eur. J. Med. Res. 1: 9-15.
- Gessler, P., Nebe, T. Birle, A., Haas, N. & W. Kachel. (1996) Neutrophil respiratory burst in term and preterm neonates without signs of infection and in those with increased levels of C-Reactive Protein. Pediatr. Res. 39: 843-848.
- Mauch L, Lun A, O'Gorman MG, Harris JS, Schulze I, Zychlinsky A, Fuchs T, Oelschlägel U, Brenner S, Kutter D, Rösen-Wolff A, Roesler J, (2007) "Chronic granulomatous disease (CGD) and complete myeloperoxidase deficiency both yield strongly reduced dihydrorhodamine 123 test signals but can be easily discerned in routine testing for CGD." Clinical chemistry 53(5): 890-896.
- Qin Y, Lu M, Gong X, (2008) "Dihydrorhodamine 123 is superior to 2,7-dichlorodihydrofluorescein diacetate and dihydrorhodamine 6G in detecting intracellular hydrogen peroxide in tumor cells." Cell biology international 32(2): 224-228.
Live Tissue Analysis of lacZ Activity .
Fluorescein di-b-D-Galactopyranoside (FDG, M0250) is a popular fluorogenic substrate for detection of cloned b-galactosidase activity (lacZ) in live cells. Enzymatic activity releases the highly fluorescent dye fluorescein (excitation at 488 nm and emission at 512 nm). Most common microscopes or microplate readers are equipped with filter sets for detection of fluorescein activity. Work from the laboratories of Sheila Nirenberg and coworkers at the Department of Physiology and Biophysics, Weill Medical College, Cornell University have defined methods for sensitive detection of lacZ activity in live cells and tissues, as well as specific ablation of these cells using the mild reducing agent 3-amino-9-ethylcarbazole [132-32-1]. Typically cells or tissues are bathed in a solution of FDG (0.5 to 2 mM concentration) for a period of time, from 30 minutes to 2 hours. Cells or tissues are then washed with buffer or media prior to observation. Cooling the cells or tissues (4oC) prior to analysis often helps prevent translocation of the dye during observation. For more information about these techniques, please see the references below or visit our website.
- Krüger A, Schirrmacher V, Khokha R, (1999) "The bacterial lacZ gene: An important tool for metastasis research and evaluation if new cancer therapies." Cancer and Metastasis 17:285-294.
- Sullivan R, Lo CW, (1997) "Histochemical and Fluorochrome-Based Detection of β-Galactosidase." Meth. Mol. Biol. 63: 229-246.
- Nirenberg S, “Photoablation of cells expressing beta-galactosidase.” Methods Mol. Biol. (Methods in molecular biology) (2000) 135: 475-80.
- Nirenberg, S., Meister, M. (1997) “The light response of retinal ganglion cells is truncated by a displaced amacrine circuit.” Neuron 18(4): 637-50.
- Nirenberg, S., Cepko, C. (1993) “Targeted ablation of diverse cell classes in the nervous system in vivo.” J. Neurosci. 13(8): 3238-51.
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