Effects of Quinones on Murine Fibroblasts
By: Susan Garcia
In the Lab of : Dr. Kristine M. Garza
BRIDGES to the Future Program
University of Texas in El Paso
July 31, 2002
The immune system is a network of players, both organs and cells, who cooperate to protect one against invasion by pathogens such as viruses, bacteria, or parasites (1). The organs of the immune system include the spleen, the thymus, the bone marrow, and lymph nodes. The cells of the immune system include neutrophils, eosinophils, monocytes, basophils, and lymphocytes. While most immune cells are generated and mature in the bone marrow, T lymphocytes, or T cells, arise in the bone marrow but mature in the thymus. Eventually they migrate and seed the spleen and lymph nodes. T cells in the spleen detect pathogens located in the blood, while T cells in the lymph nodes detect pathogens located in the tissue.
T cells are members of the adaptive immune system and are responsible for specific recognition of individual pathogens. Upon recognition of microbial components, the T cells aid in the removal of the invading pathogen. Thus, in order to protect one against all potential invading pathogens, the immune system must generate a great number of different T cells with specificities against a vast array of different microbial components. In the process of accomplishing this task, the immune system inadvertently generates T cells that recognize components of our own tissues (self). Such T cells, known as autoreactive T cells, can induce autoimmune disease. An autoimmune disease occurs when your immune system attacks your own tissue causing a clinically detectable problem (1). Some examples of autoimmune diseases include Juvenile Diabetes, Multiple Sclerosis, Lupus (SLE), and Grave's Disease. Upon recognition of self-components, the activated autoreactive T cells proliferate so that they create more copies of themselves. In normal responses against pathogens, the proliferating T cells create more T cells with specificity against the invader with the purpose of more efficiently eliminating the pathogen. However, when autoreactive T cells proliferate, they create more copies of themselves, which can cause more damage to self-tissue ultimately resulting in an autoimmune disease. How to specifically prevent autoreactive T cells from expanding and thus preventing disease is a key issue in modern immunology.
Possible chemotherapeutics include quinones. Quinones are small hydrophobic molecules that promote redox reactions by transferring electrons from one molecule to another (2). Previous studies, in tumor models, have shown that quinones have biological activity in that they induce apoptosis (3). Apoptosis is defined as "gene-directed cellular self-destruction," or programmed cell death (4). The cells die in a regulated fashion by cutting up their DNA and by “blebbing” into smaller membrane-surrounded vesicles. Another type of cell death is necrosis, however this is not the safest way for a cell to die because it causes other cells around it to die as well by releasing their intracellular fluid. There are two ways in which apoptosis can be induced. One way is by activating or turning on death receptor pathways. The other, which is believed to be the way quinones induce apoptosis, is via the mitochondrial pathway. The quinones are believed to alter the electron gradient, which drives ATP synthesis, of the mitochondria causing cytochrome C to be released and thus beginning a caspace cascade which results in apoptosis (4).
Utilizing a murine fibroblast cell line, a quinone compound, LMC4, has been tested for its ability to induce cell death and for its ability to induce apoptosis. Our approach has established a protocol by which to rapidly and effectively prescreen additional quinone compounds to be used in further testing against T cells, with hopes of ultimately testing such compounds in autoimmune disease models.
Methods & Materials
The murine fibroblasts cell line, L929, was utilized. This type of cells were used to develop the preliminary screening protocol because they grow, expand, and proliferate well in culture. They are also more easily obtainable then murine T cells. The cells were passaged twice a week at a ratio suitable for upcoming assays. The cells were maintained in DMEM media supplemented with 10% fetal calf serum, antibiotics (penicillin and streptomycin), glutamax, and b-mercaptoethanol (GIBCO/BRL – Life Technologies) at 37°C in 100% humidity and 5% CO2.
The compound LMC4, or 2,3-dimethyl-[1,4]naphthoquinone, was provided by Dr. Martinez from the Chemistry Dept. of UTEP (depicted below).
MTT Proliferation Assay
The L929 cells were seeded at 25,000 cells/well into 96-well flat bottom plates in the presence of increasing concentrations of the quinone compound. Serial dilutions of DMSO were also used as a vehicle control and cells grown alone were used as a positive control. The cells, in a total of 200 ml, were grown for 24 and 48 hrs at which time 20 ml of a 2.5 mg/ml stock of MTT (3-(4,-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium) (Sigma. St. Louis, MO) was added to each well and incubated for an additional 4 hrs. The MTT is converted to a color crystal product by mitochondrial enzymes, which are dissolved with isopropanol containing 0.1N HCl and 10% Tritan X, upon removal of the supernatant. Color development was determined spectrophotometrically on an ELISA reader at a wavelenth of 570 nm.
Propidium Iodide Staining
L929 cells were seeded at 1,000,000 cells/well into 12-well flat bottom plates and incubated with a concentration of compound that induced 50% killing as determined by the MTT assays. The cells were grown 24 and 48 hrs at which time the cells were harvested after trypsin treatment to remove the adherent L cells from the plate and were stained with 10 ml of a 100 mg/ml stock of propidium iodide (Sigma. St. Louis, MO). Binding of propidium iodide to DNA was measured by flow cytometry.
Annexin V Staining
L929 cells were seeded at 1,000,000 cells/well into 12-well flat bottom plates in the presence of compound that induced 50% death. After 24 or 48 hrs of incubation, the cells were harvested (as described), and stained with 5 ml of FITC-conjugated annexin V stock (BD Pharmingen. San Diego, CA), and assessed by flow cytometry.
The L929 cells were grown as previously described and harvested (as described previously) at 24 or 48 hrs. The cells were stained with antibody against 3’-OH groups as per manufacturer’s recommendations (Flow TACS kit) (Trevigen. Gaithersburg, MD) and assayed by flow cytometry.
Utilizing a Beckman Coulter EPICS XL flowcytometer, 10,000 events per ungated sample was collected. Data was analyzed with EXPO 32 software.
Our overall purpose was to establish a screening protocol by which to determine whether different quinone compounds induce cellular death by apoptotic mechanisms. We chose to use L929 cells, an established murine fibroblast cell line, as our indicator cell line because it grows well in culture, is easy to maintain, and is more easily obtainable than murine T cells. We first determined the number of cells to be used for the MTT assays. Figure 1 illustrates our findings. We chose to use 25,000 cells per well for future experiments, a concentration which utilized the least number of cells but provided an adequate absorbance reading.
Upon having chosen the amount of cells to work with, we next tested the ability of the quinone to kill L cells, as measured by MTT. MTT functions as a substrate for mitochondrial enzymes and is converted into color crystals. The intensity of color observed is directly proportional to the number of cells in the well. The first compound to be put to the test was LMC4. We set up an MTT assay using L cells in the presence of doubling dilutions of compound LMC4, doubling dilutions of DMSO, and media alone. The reason for using DMSO is to see how it affects the cells. Quinones are hydrophobic, therefore they are not very soluable in water and must be dissolved using DMSO. Our experiment is set up to ensure that the DMSO alone does not kill or affect the cells. L cells plus media function as a positive control for cell growth. The results are illustrated in Figure 2.
Figure 1. Determining number of L929 cells to use in 96-well plate MTT assays.
L929 cells, which were in log phase of tissue culture growth, were seeded in duplicate into 96-well flat bottom plates at 25,000 cells. The wells were serially diluted, again in duplicate, across the plate. Each dilution reduced the next wells’ cell content by half of what the previous well contained. The plates were incubated for 48 hrs at which time MTT was added and were incubated for an additional 4 hrs. Color development was assessed after soluabilization of the crystal product by spectrophotometry. Data is one of two representative experiments. Data is plotted as mean ± SEM of duplicate wells.
Figure 2. LMC4 kills L929 cells in a dose-responsive manner.
L929 cells were seeded at 25,000 cells per well into a 96-well flat bottom plate in the presence of increasing concentrations (doubling dilutions) of LMC4 (closed squares), DMSO (closed triangles), or with media alone (upside down closed triangle). The plates were incubated for 24 hrs (A) and 48 hrs (B). MTT was then added and the plates were incubated for an additional 4 hrs. Color development was determined after solubilization of the crystal product by spectrophotometry. Data is one of two representative experiments. Data is plotted as mean ± SEM of duplicate wells.
These data indicate that LMC4 causes murine fibroblasts to die. At higher concentrations, LMC4 is particularly toxic to these cells. DMSO appeared not to have much of an effect on the cells. Therefore, cell death is specifically caused by LMC4. After 48 hours, there was much more cell death than at 24 hours, suggesting that the effect of LMC4 on the cells occurs with time and is cumulative.
Before moving on to the second protocol that was to be used to determine cell death, we had to select which concentration of LMC4 to use. We did not want to choose a concentration that would be too toxic for the cells, but we also did not want to choose a concentration that was too weak. We decided to use LMC4 at a concentration of 0.03125 mg/ml, a concentration that induced approximately 50% maximal killing.
Propidium Iodide staining was our second protocol for assessing cell death. Propidium iodide is a vital dye, which binds to DNA. Only cells with compromised plasma membranes (dead or dying cells) are stained, whereas undamaged (viable) cells are not stained with this dye. Propidium Iodide is highly fluorescent upon binding to DNA, thus can be assessed using flow cytometry (3).
Figure 3 illustrates the results of this experiment. The vehicle, DMSO, does not play a part in cell death. It is apparent that the cells with LMC4 have a higher percentage of propidium iodide, demonstrating that this compound at this concentration does in fact kill murine fibroblasts. When compared to the 24 hour experiment, the 48 hour experiment shows that there were more dead cells present after a longer period of incubation.
We proceeded to determine if LMC4 induced death via an apoptotic mechanism. The first protocol we used to assess apoptosis was annexin V staining. Annexin V is a protein that detects whether the cells are inverting their cell membranes. When cells begin to apoptose, they
Figure 3. LMC4 induces death in L929 cells as measured by propidium iodide uptake.
L929 cells were seeded at 1,000,000 cells per well in a 12-well plate. The cells were incubated in the presence of 0.03125 mg/ml of compound LMC4, the concentration which induces 50% killing, or with DMSO or with media alone. The cells were incubated for 24 hrs (A) and 48 hrs (B). The cells were harvested, transferred to FACs tubes and stained with propidium iodide. The samples were immediately read by flow cytometry. Data is presented as the mean ± SEM of duplicate wells and is one of two representative experiments.
flip their phospholipids from the inner leaflet to the outer leaflet. These particular phospholipids bind to annexin V. As shown in figure 4, L cells treated with compound LMC4 had increased percentages of apoptotic cells, suggesting that LMC4 induces apoptosis. As previously observed, with time, the percentage of apoptotic or dead and dying cells increased.
Thusfar, the experiments have demonstrated convincingly that LMC4 kills murine fibroblasts. In addition, our data suggests that this quinone may be inducing death by an apoptotic mechanism. Our experiments also demonstrated that the protocols that we have used efficiently and effectively reveal quinone induction of cell death and apoptosis. There is still one more protocol to be assessed, DNA nicking. DNA nicking is a protocol used to assess whether cells die via apoptosis. This protocol will assess whether the cells are cutting up their DNA. When cells apoptose, the final outcome is that their DNA is cut up into very distinct, small pieces. Using the Flow TACS kit and flow cytometry will help us confirm if the cells are apoptosing.
There is much more to come in this research. The only compound that has been tested here is LMC4, there are still many additional quinone compounds to be tested. Once these other compounds have been tested using the protocols described here, we will proceed to test the prescreened compounds on T cells to see if they have similar affects, in hopes of ultimately identifying a potential chemotherapeutic in autoimmune disease.
Figure 4. LMC4 induces apoptotic death in L929 cells as measured by Annexin V staining.
L929 cells were seeded at 1,000,000 cells per well in a 12-well plate. The cells were incubated in the presence of 0.03125 mg/ml of compound LMC4, the concentration which induces 50% killing, or with DMSO or with media alone. The cells were incubated for 24 hrs (A) and 48 hrs (B). The cells were harvested, transferred to FACs tubes and stained FITC-conjugated Annexin V. The samples were immediately read by flow cytometry. Data is presented as the mean ± SEM of duplicate wells and is one of two representative experiments.
1 Sompayrac L. How the Immune System Works. USA: Blackwell Science, Inc.; 1999. Chapter 2, The Innate Immune System; p 17-28. Chapter 8, Immunopathology—The Immune System Gone Wrong; p 85-96.
2 Alberts B et al. Molecular Biology of the Cell. 3rd Edition. New York: Garland Publishing; 1994.
3 Rael E, Martinez L.E. Combinatorial Synthesis of Biologically Active Libraries Through Chromium-Mediated Reactions.
4 Gupta S. Molecular Steps of Death Receptor and Mitochondrial Pathways of Apoptosis. Life Sciences 2001; p 2957-2964.
Members in the lab of Dr. Garza (Karen Dearsen, etc.)
Bridges to the Future Program
National Institute of Health?