Marker Gene Monthly Newsletter   

July, 2006

Volume 6, Number 7

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Metastasis Detection using Enzymology.

Metastasis is the spread of cancer cells from a primary tumor to distant sites in the body. It can be one of the most fearsome aspects of cancer and despite significant advances in cancer detection and treatment, most deaths from cancer are due to metastasis that is resistant to conventional therapy. Minute and hidden micro-metastases often exist even in early diagnosed tumor etiology. Formation of metastases involves a series of linked, sequential steps in which tumor cells secrete tissue-destroying enzymes, which then allows them to break away from the original cancer and spead. For this reason, current drugs that only stop cancer growth may not always be able halt the metastatic spread of cancer. A number of enzymes have been linked to metastatic behavior. Among these are a variety of proteases including the matrix metalloproteases (MMPs), elastase and urokinase-type plasminogen activator (uPA) that function to degrade neighboring cell membranes and basement membranes and allow invasion of the tumor as well as initiate the metastatic process. The MMPs,also called matrixins, are a family of zinc-containing endopeptidases that play an important role in tumor metastasis and angiogenesis. At present, more than 25 different MMPs have been characterized in humans. For example, increased levels of MMP-2 (gelatinase A) expression has been demonstrated in many different human tumors.

Recent studies on breast cancer biopsies reported high levels of the enzyme, stromelysin-3 (ST-3), particularly from patients who later developed metastasis. ST-3 was found to be involved in the early steps of metastasis, which convert a tumor cell to a metastatic cell. ST-3 belongs to a well-known class of zinc-containing enzymes (matrix metalloproteinases, MMPs), but it has several unique properties. Interestingly, it does not directly break down the extracellular matrix surrounding tumor cells, but instead appears to bind to and inactivate a natural inhibitor of other tissue-destroying enzymes. This then allows these other enzymes to degrade the matrix, leading to metastasis. In addition, a tyrosine phosphatase enzyme called PRL-3 has been found in higher levels from colon cancer cells that have metastasized to the liver than in nonmetastatic colon tumors and normal colon epithelial cells. This finding suggests that an excess of the enzyme, which normally helps control intracellular activities, may somehow foster the spread of colon cancer to the liver, its principal site of metastasis. Marker Gene Technologies is working with several groups to develop and provide new and sensitive substrates for detection of these important enzymes linked to cancer metastasis. Please visit our website or see the references below for more information about these new enzyme assays.

• Nelson A.R.,Fingleton B.,Rothenberg M.L., Matrisian L.M., (2000) “Matrix metalloproteinases: biologic activity and clinical implications.” J. Clin. Oncol. 18: 1135-1149.
• Egeblad M., Werb Z., (2002) “New functions for the matrix metalloproteinases in cancer progression.” Nat Rev Cancer 2: 161-174.
• Castillo M.J., Nakajima K., Zimmerman M., Powers J.C. (1979) “ Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases.” Anal. Biochem. 99: 53-64.
• Marx, J., ( 2001) “ New Insights Into Metastasis” Science 294(5541): 281 – 282.
  

Mitochondrial Enzyme Analysis.

Mitochondria are the powerhouses of eukaryotic cells, where they can make up as much as 25% of the cell volume. Their structure varies greatly depending on cell type, cell-cycle stage or the intracellular metabolic state. The key function of mitochondria appears to be energy production through sugar metabolism, oxidative phosphorylation and lipid oxidation. Several other metabolic functions are performed by mitochondria, including ATP and urea production and heme and steroid biogenesis, as well as intracellular Ca 2+ homeostasis. Detection of the initial stages of apoptosis — programmed cell death, is often monitored by early changes in mitochondrial polarization or structural integrity. To date there remains only a partial understanding of mitochondrial function, and the mechanisms that regulate their activity in the cell.

A range of fluorescent dyes that are specific for monitoring mitochondrial function and morphology are available. Except for nonyl acridine orange (NAO) the uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Nonyl acridine orange is well retained in the mitochondria of live cells for up to 10 days, making it a useful probe for monitoring mitochondria during isolation or in cell culture. The mitochondrial uptake of NAO is reported be membrane potential independent. It apparently binds to cardiolipin in the mitochondrial membrane, but can be toxic at higher concentrations to mitochondria, regardless of their energetic state. This compound has been used to analyze mitochondria by flow cytometry (FACS) and to measure changes in mitochondrial size undergoing apoptosis.

The fluorescent probe JC-1 (5,5',6,6'-tetrachloro-1,1',3 ,3'-tetraethylbenzimidazolylcar bocyanine iodide), is another common mitochondrial labeling dye. It exists as a monomer with green fluorescence at low concentrations or at low membrane potential. But at higher concentrations (> 0.1 uM) or at higher potentials, JC-1 forms so-called red-fluorescent "J-aggregates" that change to a broader excitation spectrum and an emission maximum at approximately 590 nm. Therefore the emission of JC-1 can be used to measure mitochondrial membrane potential by monitoring the ratio of the signals from the green-fluorescent JC-1 monomer (EX514/EM529 nm) and the red-fluorescent J-aggregate (EM 590 nm). The ratio of red-to-green JC-1 fluorescence seems to be independent of mitochondrial size, shape and density. But in apoptosis, mitochondrial depolarization can easily be monitored using this same green-red fluorescence ratio (red = normal; green = apoptotic or necrotic). Usual fluorescein and tetramethylrhodamine filter sets can be used for measuring monomer and J-aggregate forms, respectively. Both forms can be observed simultaneously using a fluorescein longpass optical filter set.

Rhodamine 123 ( M0542 ) is a cell-permeant, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxicity. Uptake and equilibration of rhodamine 123 is rapid (a few minutes) compared with dyes such as DASPMI (4-Di-1-ASP) which may take 30 minutes or longer. Viewed through a fluorescein longpass optical filter, fluorescence of the mitochondria of cells stained by rhodamine 123 appears yellow-green. Viewed through a tetramethylrhodamine longpass optical filter, however, these same mitochondria appear red. Unlike the lipophilic rhodamine and carbocyanine dyes, rhodamine 123 apparently does not stain the endoplasmic reticulum. Dihydrorhodamine 123 ( M0545 ) is a reduced form of the same dye that is oxidized to the fluorescent product Rhodamine 123 by intracellular reactive oxygen species and then stains mitochondria. However, the oxidation may occur in organelles other than the mitochondria. For more information about these products and their assays in mitochondria, please see our website or the reference below.

• Frey T.G., Mannella C.A. (2000) "The internal structure of mitochondria." Trends Biochem. Sci. 25: 319-324
• Reers M., Smith T.W., Chen L.B., (1991)"J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential” Biochemistry 30: 4480-4486.
• Johnson L.V., Walsh M.L., Chen L.B., (1980) "Localization of mitochondria in living cells with rhodamine 123." Proc. Natl. Acad. Sci. U S A 77: 990-994.
• Summerhayes I.C., Lampidis T.J., Bernal S.D., Nadakavukaren J.J., Nadakavukaren K.K., Shepherd E.L., Chen L.B. (1982) “Unusual Retention of Rhodamine 123 by Mitochondria in Muscle and Carcinoma Cells” Proc. Natl. Acad. Sci. USA 79(17): 5292-5296.
• Chen LB. (1989) "Fluorescent labeling of mitochondria." Methods Cell Biol. 29: 103-123.
• Septinus M., Berthold T., Naujok A., Zimmermann H.W., (1985) "Hydrophobic acridine dyes for fluorescent staining of mitochondria in living cells. 3. Specific accumulation of the fluorescent dye NAO on the mitochondrial membranes in HeLa cells by hydrophobic interaction. Depression of respiratory activity, changes in the ultrastructure of mitochondria due to NAO. Increase of fluorescence in vital stained mitochondria in situ by irradiation." Histochemistry 82: 51-66.

New Long-Wavelength Lipase Substrate and Kit.

Lipases are a family of enzymes that release fatty acids from triacylglycerols, glycolipids and lipoproteins in a site specific manner. Most lipases have optimum activity for the primary ester groups of triglycerides, while some lipases remove fatty acyl groups from either the C-1 or C-3 acyl positions of triacylglycerols or from glycolipids or lipoproteins. The substrate is typically not a single molecule, but a nonaqueous phase of aggregated lipid. Lipase activity, ubiquitous among most cells, can be monitored using our new broad spectrum, long wavelength fluorescent substrate, resorufin oleate (Product No. M1214-002), contained in the MarkerGene ™ Long Wavelength Fluorescent Lipase Assay Kit ( M1214 ). Upon enzymatic cleavage, the red fluorescent compound, resorufin ( M0202 ; EX 571nm, EM 587nm), is released and activity measurements are easily obtained either in vitro , in cell lysate preparations, or in vivo . This new kit contains enough substrate for numerous assays and control experiments, and also contains reference standards and a detailed protocol for use. It is especially useful for those situations where a second analyte is being measured at a different wavelength using a fluorescein (green) or coumarin (blue) based substrate, since multiplexing of the two signals can be used to monitor both activities simultaneously. Please see the references below or our website for more information and applications of this new substrate and reagent.

• Dousset, N., Negre, A., Salvayre, R., Rogalle, P., Dang, Q.Q., Douste-Blazy, L. (1988). “Use of a fluorescent radio labeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase.” Lipids 23: 605-608.
• Liodakis, A., Drew, J., Chan, R., Sawyer, W.H., (1991) “Spectrofluorometric determination of lipase activity.” Biochem. International 23(5): 825-834.
• Main, L.A., Okumura-Noji, K., Ohnishi, T., Yokoyama, S. (1998). “Cholesterol ester transfer protein reaction between plasma lipoproteins.” J Biochem (Tokyo) 124: 237-243.
• Negre, A., Salvayre, R., Dousset, N., Rogalle, P., Quan Dang, Q., Douste-Blazy, L. (1988) “Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice.” Biochim Biophys Acta 963: 340-348.
• Rosseneu, M., Taveirne, M., Caster, H., Van Biervliet, J. (1985). “Hydrolysis of very low-density lipoproteins labeled with a fluorescent triaglycerol: 1,3-dioleoyl-2-4(pyrenylbutanoy l)glycerol.” Eur J Biochem 152: 195-198. D

Luciferase Containing Vectors for analysis of Transcription Factors.

Eukaryotic gene expression is regulated by a wide variety of developmental and environmental stimuli. Among the most important intracellular methods of control are transcription factors that bind to the promoter or enhancer regions of genes and activate expression. A transcription factor is a protein that regulates the activation of transcription in the eukaryotic nucleus. Transcription factors localize to regions of promoter and enhancer sequence elements either through direct binding to DNA or through binding other DNA-bound proteins. They act by promoting the formation of the preinitiation complex (PIC) that recruits and activates RNA polymerase. The regulation of transcription factors is a highly complex process as it is dependent upon a number of events, most notable of which are the presence of other DNA binding proteins (including other transcription factors) as well as local chromatin structure.

This process begins when an extracellular signaling molecule binds to a specific intracellular receptor. The extracellular signal is then transmitted through a series of molecular cascades that activate or deactivate specific transcription factors. These factors then regulate gene expression. The expression of any given gene is controlled by multiple transcription factors, which in turn are modulated by multiple signal transduction pathways. These pathways can also be organized into networks. In order to monitor transcription factor activities and measure their quantitative levels inside living cells, researchers at Panomics, Inc. ( www.panomics.com ) have developed a series of luciferase containing vectors that contain cis-acting DNA transcription factor binding domains inserted in the promoter region of plasmid vectors. When cells are transfected with these individual vectors, if a transcription factor is active, it will bind to the vector and initiate luciferase production, which in turn can be monitored using the light emission from action upon its substrate D-Luciferin ( M0237 , M0626 ). The combined system provides an elegant way of monitoring specific transcription factors in vivo. To date over 50 transcription factors have been developed, and are available commercially. For more information about these new transcription factor assay systems, please visit their website or see the references below.

• Müller S., Kammerbauer C., Simons U., Shibagaki N., Li LJ ; Caughman S.W., Degitz K., (1995) “Transcriptional regulation of intercellular adhesion molecule-1: PMA-induction is mediated by NF kappa B.” J. Invest. Dermatol. 104: 970–975.
• Orzechowski H.D., G?nther A., Menzel S., Zimmermann A., Funke-Kaiser H., Real R., Subkowski T., Zollmann F.S., Paul M.,  (2001) Transcriptional mechanism of protein kinase C-induced isoform-specific expression of the gene for endothelin-converting enzyme-1 in human endothelial cells. Mol Pharmacol 60: 1332–1342.
• You, L ., Jakowlew S., (1997) “Identification of early growth response gene-1 (Egr-1) as a phorbol myristate acetate-induced gene in lung cancer cells by differential mRNA display.” Am. J. Respir. Cell Mol. Biol . 17: 617–624.
• Eyries, M., Agrapart M., Alonso A., Soubrier F.,  (2002) “Phorbol ester induction of angiotensin-converting enzyme transcription is mediated by Egr-1 and AP-1 in human endothelial cells via ERK1/2 pathway.“ Circ. Res 91: 899–906.
• Gttlicher, M., et a l. (1998) J Mol Med 76:480–489.
• Frønsda, K., et. a l. (1998) J Biol Chem 273: 31853–31859.
  
  

Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you.