Marker Gene Monthly Newsletter - Volume 11, Number 5 - May, 2011

Luciferase for Monitoring Mitochondrial Activity

The mitochondria are one of the main sources of ATP generation in the cell. Since ATP is the major source of high-energy phosphate bonds within the cell, monitoring mitochondrial ATP activity can therefore be useful in analysis of cellular and mitochondrial metabolism as well as overall cell health and viability. Targeted luciferases have been developed for various organelles within the living cell which are able to report on the localized ATP levels within their respective compartments. Recently researchers have utilized cloned luciferase activity to monitor ATP generated intracellularly in mitochondria by adding a targeting sequence to the luciferase protein that binds it to the outer surface or mitochondrial matrix of the mitochondria. By appending on the N-terminus of the luciferase protein the targeting sequence for the COX8 protein, several studies have been able to monitor mitochondrial disorders or the effects of cofactors (e.g. calcium) by sensitive analysis of ATP generation.

The firefly luciferase enzyme utilizes the substrates D-Luciferin and ATP with CoA and Mg⁺² ion to produce light. But if the other cofactors are present, the protein can also be used to measure ATP levels even at very low levels. Light output can be quantitated from single living cells after the addition of the relatively cell-permeant substrate D-Luciferin (M0237) or more permeant substrates such as D-Luciferin, ethyl ester (M0906). Under most conditions, oxygen and other cofactors other than ATP are not limiting. Furthermore, the amount of ATP utilized by the luciferase reaction has been found to represent only a tiny fraction of the total cellular ATP and will be essentially non-perturbing for normal cellular ATP homeostasis. Studies in which both cytosolic and mitochondrial targeted luciferase activities were monitored simultaneously could be used to differentiate mitochondrial versus background effects on ATP homeostasis. For more information about these techniques, please see the references below or visit our website.

• Bell CJ, Manfredi G, Griffiths EJ, Rutter GA (2006) "Luciferase Expression for ATP Imaging: Application to Cardiac Myocytes." Meth. Cell Biol. 90:341
• Gajewski CD, Yang Y, Schon EA, Manfredi G (2003). "New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters." Mol. Biol. Cell 14(9): 3628-3635.
• Jouaville LA, Pinton P, Bastianutto C, Rutter GA, Rizzuto R (1999) " Regulation of mitochondrial ATP synthesis by calcium: Evidence for a long-term metabolic priming." Proc. Natl. Acad. Sci. USA 96(24): 13807-13812.
• Porcelli AM, Pintonfi P, Ainscowfi EK, Chiesa A, Rugolo M, Rutter GA Rizzuto R (2001) "Targeting of Reporter Molecules to Mitochondria to Measure Calcium, ATP and pH." Meth. Cell Biol. 65: 353-380.
  

Suppression of Cancer Growth in Several Cancer Cell Lines

Cancer remains one of the nations leading health concerns and has caused countless resources to be spent on research into its etiology and treatment. Current cancer treatments include surgery, radiation, and chemotherapy which often utilize targeted inhibition of tumor growth mechanisms.  Recent work from the laboratory of Evangelos D. Michelakis and coworkers at The University of Alberta have investigated a new and simple class of tumor inhibitors with promising results. Dichloroacetate (DCA), an analogue of acetic acid, has been found to reduce cancer cell growth by promoting apoptosis, decreasing proliferation, and inhibiting tumor expansion. In the cell, the pyruvate dehydrogenase (PDH) cycle converts cytoplasmic pyruvate to acetyl-CoA, which is used to drive ATP synthesis. In this process, the mitochondrial membrane acquires a negative potential through the efflux of H+ across the membrane (ΔΨm). Cancer cells often have a hyperpolarized mitochondrial membrane in which the PDH cycle is inhibited by pyruvate dehydrogenase kinase (PDK).  It was previously thought that this was a result, and not a potential cause of uncontrollable growth of cancer cells.

The current studies used DCA to inhibit PDK in order to restore membrane potential and shift the metabolic state from glycolysis to glucose oxidation. The membrane potential was restored through the increase of reactive oxygen species, which are known to dilate certain ion channels to uphold the mitochondrial potential. It was found that the K+ channels were expressed with the production of ROS. The effects caused reduced proliferation, promoted apoptosis, and inhibited tumor growth without any effect on normal cells. The ROS activity of the cancer cells as well as the mitochondrial membrane potential were measured in these studies.  To find out more about ROS assay kits and mitochondrial membrane potential measurements, please see references below or visit our website.

• Bowker-Kinley MM, Davis WI, Wu P, Harris RA, and Popov KM (1998) “Evidence for existence of tissue-specific regulation of the mammalian pyruvate dehydrogenase complex.” Biochem. J.  329: 191–196.
• Bonnet S, Archer SL, Allalunis-Turner J, Haromy A, Beaulieu C, Thompson R, Lee CT,  Lopaschuk GD, Puttagunta L, Harry G, Hashimoto K, Porter CJ, Andrade MA, Thebaud B, Michelakis ED (2007) “A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth.” Cancer Cell. 11:37-51.
• Koukourakis MI, Giatromanolaki A, Sivridis E, Gatter KC, Harris AL (2005) “Pyruvate dehydrogenase and pyruvate dehydrogenase kinase expression in non small cell lung cancer and tumor-associated stroma.” Neoplasia 7: 1–6.
• Stacpoole, PW, Kerr DS, Barnes C, Bunch ST, Carney PR, Fennell EM, Felitsyn NM, Gilmore RL, Greer M, Henderson GN, Hutson DN, Neiberger RE, O'Brien RG, Perkins LA, Quisling RG, Shroads AL, Shuster JJ, Silverstein JH, Theriaque DW, Valenstein E (2006) “Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children.” Pediatrics 117: 1519– 1531.
• Wang Z (2004) “Roles of K+ channels in regulating tumour cell proliferation and apoptosis.” Pflugers Arch. 448: 274–286.

Fluorescent Oligonucleotide Labeling for Southern Blot Analyses

Southern blotting is a commonly used method to detect the presence and quantity of a specific DNA sequence in a DNA sample, often using genomic DNA. The DNA is first digested by restriction enzymes, then separated on an agarose electrophoresis gel. After the gel has run the DNA fragments are then blotted onto a nitrocellulose membrane and oligonucleotide probes specific to the target are applied to the membrane. These probes are labeled, often radioactively, to allow the detection of the target sequence. This typically requires exposure of the blot membrane to X-ray film. Because the use of radiolabeling has obvious safety concerns fluorescently labeled probes have become a preferable method for such analyses, with the signal being similarly detected using UV light and photographic analysis.
Marker Gene's new OliGlo™ kits offer a simple and quick method of labeling probes for Southern Blot analysis. These kits utilize a direct labeling methodology through reaction with the phosphate groups (terminal and backbone) on oligonucleotide, DNA or RNA samples, leaving the base pairing intact for hybridization. Moreover, the active labeling reagents are prepared in situ from stable precursor molecules derived from a variety of highly fluorescent dyes or hapten labels, allowing the highly reactive labels to function at optimum efficiency for each sample. The OliGlo™ kits allow molecular biologists and clinical researchers to label Southern blot probes for easy detection and quantification. The currently available Universal Labeling Kits can be utilized for such ultrasensitive Southern Blot analysis. Marker Gene also offers several FISH kits for use in Fluorescence In-Situ Hybridization Methods as well. To find out more about these methods, please see the references below or visit our website.

• Forster AC, McInnes JL, Skingle DC, Symons RH. (1985) "Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin." Nucleic Acids Res. 13: 745-791.
• Kessler C. (1995) "Methods for nonradioactive labeling of nucleic acids." in Nonisotopic probing, blotting, and sequencing. (Kricka L J, Ed.) pp 41-109, Academic Press, San Diego.
• Olejnik J, Krymanska-Olejnik E, Rothschild KJ. (1998) "Photocleavable aminotag phosphoramidites for 5'-termini DNA/RNA labeling." Nucleic Acids Res. 26(15): 3572-3576.
• Rihn H, Coulais C, Bottin MC, Martinet N. (1995) "Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods." Biochem. Biophys. Methods 30: 91-102.
• Southern EM. (1975) "Detection of specific sequences among DNA fragments separated by gel electrophoresis". J Mol Biol. 98(3): 503-17

Mitochondrial Membrane Potential

Fluorescent dyes that accumulate in the mitochondria can be used to assess mitochondrial activity and function. Measurement of the distribution of dye in the cytosol versus the mitochondia has been used to determine mitochondrial membrane potential (ΔΨm) using the Nernst equation. Among the dyes useful for these measurements are tetramethylrhodamine esters. In particular tetramethylrhodamine, ethyl ester (TMRE, M1722) has found significant utility in these applications. TMRE is a positively charged lipophilic fluorophore with red-orange fluorescence (EX 549; EM 575 nm) that rapidly accumulates in the mitochondria due to the relative negative charge of active mitochondria with respect to the cytosol. TMRE can also be used to monitor spontaneous transient depolarizations of ΔΨm, sometimes called mitochondrial flickers most commonly attributed to transient activation of the mitochondrial permeability transition pore (PTP). TMRE can therefore serve as a “Nernstian” indicator of ΔΨm, with mitochondrial depolarization resulting in a loss of the dye from the mitochondria and a decrease in mitochondrial fluorescence intensity. Cells are typically bathed in HBS buffer containing the dye at 25 - 100 nM concentration for 15 min. for staining. Marker Gene now offers highly purified TMRE for your mitochondrial activity studies. For more information about these assays and reagents please see the references below or visit our website.

• O'Reilly CM, Fogarty KE, Drummond RM, Tuft RA, Walsh JV (2003) "Quantitative Analysis of Spontaneous Mitochondrial Depolarizations." Biophys. J. 85(5): 3350–3357.
• Collins TJ, Berridge MJ, Lipp P, Bootman MD (2002) "Mitochondria are morphologically and functionally heterogeneous within cells." EMBO J. 21(7): 1616-1627.
• Duchen MR, Leyssens A, Crompton M (1998) "Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes." J. Cell Biol. 142(4): 975-988.

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