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Cho, Liu 1
The Detection of Differences Between Normal and Cancerous Stem Cells Through Analysis of Morphology, Gene Expression, and Effects of Dichloroacetate Hyunjii Cho and Jimmy Liu, advisers: Marie Olzewski, and Dr. Morris Kletzel at the Children’s Memorial Hospital Stem Cell Laboratory Abstract Developments in oncology research point to stem cells as a promising, new means through which to cure cancer. We observed the morphology, gene expression, and response to dichloroacetate (DCA) of normal and cancerous stem cells. Mononuclear cells isolated from human blood were cultured for experimentation. We tested several different DCA concentrations to determine if cancer cells and normal cells die in DCA. To determine which stem cell markers exist on the cells, we performed flow-cytometry, a technique used to analyze physical and chemical characteristics of single cells flowing through an electronic detection apparatus. We also used RT-PCR to determine the genes expressed. When DCA was applied to the experimental group, the concentration of cells was successfully suppressed, implying that DCA has the effect of prohibiting cell proliferation. This implies the restoration of apoptosis in cancerous cells and the preservation of normal cells. DCA could be pursued as a cancer therapy because of its preferential effect on cancerous cells, and consequently, we plan to further test its potential. In particular, we plan to test DCA on cell cultures with both normal and cancerous cells combined; current tests have only tested DCA on normal and cancerous cells individually. Focusing Question What are the effects of dichloroacetate on normal and cancerous stem cells with respect to cell concentration and gene expression? Introduction We decided to research oncology because of its significance in today’s society. More specifically, we focused on the elucidation of the differences between cancerous and normal adult stem cells by analyzing key characteristics such as morphology with
Cho, Liu 2 stem cell markers and gene expression, because we firmly believe that it will be essential in discovering the mechanisms through which cancer progresses. Another goal of our experimentation was to test the efficacy of dichloroacetate (DCA) as a targeted cancer therapy. DCA is a promising compound in cancer research because of its nontoxicity and versatility. The mechanism through which it acts is unique: it exploits the dysfunctional mitochondria, a characteristic unique to cancer cells. Because of this, as well as its small size, it is able to treat various types of cancer, including brain, lung and breast cancers. DCA restores apoptosis in cancer cells while leaving normal cells unaffected. The applications of dichloroacetate as a novel cancer therapy are limitless, because DCA has been previously used to treat rare metabolic disorders; it is known to be relatively safe and non toxic. Our study further tests the compound in the context of the immortalized human myeloid leukemia cell line K562. Previous research has been conducted with DCA testing its effectiveness on normal and cancerous cells, but our experimentation adopts a novel approach through analyzing the effects of DCA on normal and cancerous stem cells (Bonnet, Archer, Allalunis-Turner, & Haromy, 2007). Hematopoietic stem cells are isolated from the peripheral blood or bone marrow and are capable of selfrenewal and differentiation into specialized cells. Apoptosis, programmed cell death, is necessary in order to prevent an excessive number of cells. Normal apoptotic functions are not present in cancerous stem cells, allowing unlimited cell regeneration. In healthy stem cell division, the cell regenerates itself and also creates a progenitor cell. Although the progenitor cell cannot regenerate itself, it is capable of dividing indefinitely into mature specialized cells (Lee & Herlyn, 2007). The progenitor cell then matures and become a specialized cell. Stem cells are characterized by both “selfrenewal and their ability to produce cells that differentiate” (Morrison & Kimble, 2006), called progenitor cells. Stem cells divide using two strategies: asymmetric and symmetric stemcell divisions. In
Cho, Liu 3 asymmetric stemcell division, stem cells divide to produce one identical daughter of itself, and one daughter that is capable of differentiating. Through this process, stem cells are unable to expand in number and thereby unable to produce stemcell pools that are needed for development and for cell regeneration after injury. Therefore, there must be another method that stem cells utilize to maintain control of their numbers. “Symmetric divisions are defined as the generation of daughter cells that are destined to acquire the same fate.” (Morrison et al., 2006). Through symmetric division, stem cells are able to divide into two stem cell daughter cells or two progenitor cells. Most stem cells are able to divide both asymmetrically and symmetrically, and these two modes are controlled by developmental and environmental signals. However, this balance is sometimes disrupted and defective in disease states.
Division of a Stem Cell
Source: University of Medicine and Dentistry at New Jersey (2007). Figure 1. Division of stem cells displaying selfrenewal and the progenitor cell. Stem cell matures into a progenitor cell, and differentiates into various other cells.
Cho, Liu 4 The K562 human leukemia cell line is strikingly similar to stem cells. According to Young and Hwangchen, K562 has characteristics of selfrenewal and pluripotency similar to those of stem cells (1981, p.7073). Comparable to leukemic stem cells, K562 also expresses the WT1 gene (Hidehiko, Masato, & Etsuko, 2006). According to Kanato, Hosen, and Yanagihara, WT1 expression is restricted to hematopoietic stem cells or progenitor cells. The Wilm’s Tumor 1 gene (WT1) is overexpressed in leukemia, thus explaining the proliferation of blood cells causing leukemia (2005). In normal subjects, there is low or undetectable expression of the WT1 gene; however, this gene is widely expressed in leukemic cell lines, as well as in the majority of lymphoid and myeloid leukemias of childhood and adulthood (HernandezCaballero, Mayani, & Montesinos, 2007). The rationale for testing the efficacy of DCA on stem cells is the methods of current chemotherapy and its success rate. Recent research in oncology points to stem cells as a novel method through which to study cancer. When a tumor is targeted, often there is a stem cell that differentiates into progenitors. The tumor is essentially composed of cancerous cells surrounding a few cancerous stem cells. The reason that many patients come out of remission is that although the cancer cells are eradicated, the cancerous stem cells remain viable (Lee & Herlyn, 2007). This poses a problem, as cancerous stem cells have unlimited potential to differentiate, thus causing cancer to relapse (Lee & Herlyn, 2007). A secondary method used to measure the efficacy of DCA is the analysis of the expression of the Wilm’s Tumor gene (WT1). The presence of this gene indicates tumor progression, which requires excessive cell proliferation (Kanato, Hosen, Yanagihara, Nakagata, Shirakata, & Nakasawa, 2005). If WT1 expression is not detected, then the culture of cells will not proliferate rapidly and is not cancerous.
Cho, Liu 5 In a previous study at the University of Alberta, it was discovered that DCA normalizes a dysfunctional mitochondria, thus restoring apoptotic function (Bonnet et al., 2007). More specifically, cancer is unique in that it has a high membrane potential, downregulated K+ channels, and metabolizes through glycolysis. Membrane potential is the difference in electrical voltage between the inside and outside of the cell membrane (Alberts, 2002). Regular membrane potential allows for a stable cell environment; DCA was recently discovered to decrease the high mitochondria membrane potential. In addition, DCA upregulates the K+ channels and shifts metabolic activity from the cytoplasmbased glycolysis to the mitochondriabased glucose oxidation (Bonnet et al., 2007). By testing the efficacy of DCA on stem cells, we hope to further confirm the efficacy of DCA. We hypothesized that DCA would have a positive effect in restoring normal apoptosis, or programmed cell death, in cancer stem cells, and effectively inhibit cell proliferation. We also hypothesized that DCA would leave normal stem cells unaffected. Materials and Methods: In order to proceed with the experiment, blood samples that were ready to be discarded from the Children’s Memorial Hospital were obtained. K562 cells were used in place of cancer stem cells due to their similarities: K562 cells, like cancer stem cells, are able to self-renew and proliferate, as well as express the Wilm’s Tumor 1 (WT1) gene, which is exhibited in stem cells. Mononuclear cells, or stem cells were isolated by creating a density gradient. Before diluting the blood, an initial cell count was taken with the Cell Dyn 1700 machine. We then created a density gradient with the use of FicollPaque, underlayering blood with Ficoll-Paque. To keep the sample sterile, we avoided contact between the Ficoll-Paque bottle and the test tube when adding the Ficoll-Paque. Blood diluted with phosphate buffered saline (PBS) in a ratio of 1:1 and was added to this test tube. The test tube was balanced in a centrifuge and spun at 3000 rpm for 10 minutes
Cho, Liu 6 without a brake. Mononuclear cells were removed from the resulting contents, as shown in Figure 2.
Figure 2. Density gradient using Ficoll-paque.
These cells were then collected and washed with PBS and spun in the centrifuge for 5 minutes at 3000 rpm. Afterwards, the PBS and plasma were poured out and the compacted cell pellet remained at the bottom (Figure 3).
Cho, Liu 7
Figure 3. Pellet at the bottom of a test tube formed from the accumulation of mononuclear cells.
Approximately 2 mL of RPMI tissue culture was added in preparation of culturing the cells. The cell pellet was resuspended and mixed well. A final cell count was taken with the Cell Dyn 1700 in order to calculate the amount of cells per well would be needed. In our experiment, we used about 1.0x106-2.0x106 cells per well for normal bone marrow stem cells, while we used about 0.1 x 106 cells for K562, since this human immortalized myelogenous leukemia cell line proliferates rapidly. In order to culture these cells, 1mL of RPMI was added to each well and the plate was placed in the incubator. The RPMI was changed roughly every week for normal bone marrow stem cells, and about every 3 days for the K562 cell cultures. Cell viability was tested with Trypan Blue. 10 µL of the cell sample was mixed with 20 µL of Trypan Blue. Then this mixture was inserted into a sterilized hemacytometer; the hemacytometer was sterilized to remove any residue before examining the sample of cells and to avoid contamination. The cells were viewed under a microscope and viable and dead cells were counted. This method works because when a cell is not viable, the function of its semi-permeable membrane is compromised. This allows the penetration of Trypan Blue into the cell; unviable cells are permeated with
Cho, Liu 8 Trypan Blue, while the semi-permeable membrane of living cells prevents the penetration of Trypan Blue; the cell is not blue. For our DCA experiment, we made 5, 10, 20, and 40 mM concentrations of DCA in RPMI solution. To the 1 mL solution of cell culture we added 1 mL of the DCA solution. Mixing the DCA solution to another 1 mL solution diluted the concentration of DCA by half. Thus actual 5, 10, 20, and 40 mM concentrations of DCA became 2.5, 5, 10, and 20 mM concentrations, respectively. DCA was added to the cells after a few days of culturing them. Cell concentrations of the cells in DCA were measured after 48 hours for normal stem cells, and 96 and 168 hrs for K562 cells. We also used reverse transcriptase-PCR to analyze the effect of DCA on the K562. Total RNA was extracted using QIAamp RNA Blood mini-kit (QIAGEN, Inc., Valencia, CA, USA). A 2-step reverse transcriptase (RT)-PCR in a 20-uL reaction volume with 1 microgram (ug) of total RNA from each sample was conducted. The reaction buffer included 4 uL of 25 nM MgCl2, 8 uL of dNTPs, 1 uL of Oligo d(T)16, 2 uL of 10xPCR buffer II, 1 uL of RNase inhibitor, and 1 uL of Moloney Murine Leukemia Virus RT (Applie Biosystem, Foster City, CA, USA). We used primer pairs that were designed to locate particular nucleotide fragments for mRNA of the WT1 gene. Reverse transcription was incubated at 42 degrees Celsius in a water bath for 60 minutes. The it was denatured at 96 degrees Celsius in a thermal cycler for 10 minutes. 10 microliters of the complementary DNA was amplified during the 1st round of PCR. There was an initial 30 cycles of denaturation for 5 minutes, 5 seconds at 95 degrees Celsius amplification, annealing at 55 degrees Celsius fir 5 seconds, and extension at 72 degrees Celsius for 10 seconds. A second round was performed by taking 1 uL of the 1st round of amplified product and reamplifying with inner primers utilizing the LightCycler System with SYBR-Green 1 RNA master mix reagent (Roche Diagnostics, Biochemica, Indianapolis, IN, USA). Once PCR was complete, the LightCycler Software calculated the concentration of target molecules. Using a 10-fold serial dilution of K562 leukemia cell line and the Bone Marrow 2 (BM2) sample, a standard curve was generated for each sample. Values of 1x 100ng/uL and higher were considered WT1 positive. PCR products
Cho, Liu 9 were then run through a 1.5% agarose gel electrophoresis. Finally the data was analyzed in the form of graphs.
Results Figure 4 displays the increase in K562 cell concentration as time increases, thus confirming the indefinite proliferation of K562. The initial cell concentrations of K562 at Day 0 were 0.1 thousand per microliter (K/uL). K562 Control (Jan. 16.08- Jan. 30. 08) 3.5
1.0877x
y = 0.0385e 2 R = 0.9541
3
Concentration (K/uL)
2.5
2
1.5
1
0.5
0 D0
D3
D10
D14
Time (days)
Figure 4. Bar graph of the cell concentration as time increases. K562 was cultured and the cell concentration of the controls (without DCA) were recorded at various times in a 14 day period. The graph in figure 5 displays a decrease in cell concentration of K562 as the DCA concentration increases.
Cho, Liu 10
Figure 5. Protocol a: The effects of increasing DCA concentration on the cell concentration of K562. Cell concentration (K/uL) of K562 cell cultures with no DCA and cultures with 2.5 mM, 5mM, 10mM, and 20mM were observed after 168 hours after the addition of DCA. The highest concetrations of DCA from protocol a were tested again in another set of experiments, protocol b. The resulting K562 cell concentrations in 10 mM and 20mM DCA after 96 hours of the addition of DCA shows to be far less than that of the control measured at the same time.
Cho, Liu 11 Figure 6. Protocol b: K562 cell concentrations on day 14 of the culture and 96 hours after adding DCA to certain cultures. 10mM and 20mM DCA concentrations were tested and compared to the day 14 control culture. Protocol c consisted of Bone Marrow Sample #2 (BM2) normal bone marrow stem cell cultures at different concentrations. The effects of the various DCA concentrations on BM2 were relatively similar. At day 0 of the culture, the initial cell concentration was 2.0K/uL. At day 16 of the culture and 48 hours after the addition of DCA to some of the wells of the culture, the sample was observed. The concentrations were relatively around 1.7K/uL. There were no significant differences between the BM2 cell concentration of the control and the DCA-treated cultures. Protocol c: BM2 March 7 Day 16 (48Hrs in DCA) 2 1.8 1.6
1.72
1.7
Control
5mM
1.72
1.7333333
10mM
20mM
Cell Concentration (K/uL)
1.4 1.2 1 0.8 0.6 0.4 0.2 0 DCA Concentration (mM)
Figure 7. Protocol c: BM2, a normal stem cell sample, was treated with various concentrations of DCA. The resulting cell concentrations after 48 hrs of immersion in DCA were compared to the control, which had been cultured for a total of 14 days. A secondary result of experimentation is the analysis of gene expression. Again, the most significant difference can be evidenced through a comparison of non-DCA treated cultures and DCA-treated cultures. As shown in Figure 8 and Figure 9, when a pristine culture of K562 cells is used as the baseline, the two cultures treated with 20 mM of DCA from both protocol a and b have low expressions of WT1, the cell proliferation gene. BM2, the normal stem cell sample, also has a very low, almost undetectable, expression of WT1. The control K562 samples and the 10mM DCA concentration K562
Cho, Liu 12 samples all express the WT1 relatively higher than the 20mM DCA treated K562 samples.
Figure 8. WT1 expression in K562 controls, 10mM and 20mM DCA treated K562 samples, and BM2. The resulting data was examined through the LightCycler Data Analysis.
Figure 9. WT1 expression show in bar graphs for K562 controls, 10mM and 20mM DCA treated K562 samples, and BM2.
Conclusion
Cho, Liu 13 In the first set of experiments (protocol A), various concentrations of DCA were tested on cultures of 2x106 cells per well: K562 control group (n=2), K562 group with 2.5mM (n=2), 5mM DCA (n=2), K562 group with 10mM DCA (n=2). The control group, which was not treated with DCA, continued normal cell proliferation. The DCA-treated groups exhibited a decreased concentration of cells, indicating decreased cell proliferation. Using regression analysis, we determined that the concentration of cells decreased exponentially as the concentration of DCA increased. The second set of experiments (protocol B) consisted of testing 10mM and 20mM DCA concentrations on K562. 10mM and 20mM DCA concentrations were chosen for the second set of experiments (protocol B) on K562 because they exhibited almost no increase in cell concentration. The initial cell concentration was 0.1 K/uL, while the cell concentrations of K562 cells in 10mM and 20mM DCA for 168 hrs only had 0.2 K/uL cell concentrations. This is not a significant increase and can be accounted for by few trials, and human and machine (Cell Dyn 1700) error. Protocol B confirmed that DCA may inhibit cell proliferation because it also showed that cell concentrations did not increase significantly from the initial cell concentration of 0.1 K/uL and they also did not proliferate as much as the control did. RT-PCR was performed on protocol A and protocol B. The expression of WT1 in protocol A and B was measured through RT-PCR. In protocol A, the K562 group treated with 10mM of DCA showed that the cells still expressed the Wilm’s tumor suppressor gene (WT1), which regulates cell proliferation. However, the cells in protocol A that were treated with 20mM of DCA did not significantly exhibit the WT1 gene. The test was repeated for protocol B and similar results were achieved. These results indicate that a 20mM concentration of DCA is more effective than a 10mM concentration because it successfully inhibits the expression of WT1. They also support the correlation between increased DCA concentration and decreased cell concentration. The effects of DCA were also tested on normal, non-cancerous adult stem cells (Sample BM2). DCA had no significant effect on the cell concentrations of BM2. This shows that while DCA affects cancerous cells, it does not affect normal stem cells. More
Cho, Liu 14 tests will need to be performed on normal cells in the future, in order to wholly support our conclusion. Due to the lack of normal bone marrow stem cells available, we were not able to perform many tests on normal stem cells. Our findings in the morphology through analysis of stem cell markers contradict the discoveries in WT1 expression, and were thus, unexpected. Through flow cytometry, we found that the percentage of K562 cells that are both CD123 and CD 34 positive increases as the concentration of DCA increases. This means that the percentage of K562 cells that are capable of proliferating have a positive correlation with DCA concentrations. The stem cell marker CD123 may be present on cells for more than just capability of proliferation; therefore, the RT-PCR results would prove to be more reliable. Gene expression is more specific in determining function than the stem cell markers. In order to understand why the two assays contradict each other, further research will be conducted attempting to determine the cause of the presence of CD123 on cells. One might hypothesize that the CD123 stem cell marker has other functions than indicating cell proliferation. The prospect of dichloroacetate as a novel, therapeutic cancer treatment is realistic because it restores mitochondrial function, one of the fundamental pathways unique to the progression of cancer. Additionally, as it has been used for many decades in the treatment of metabolic diseases, it is known to be relatively non-toxic (Bonnet, et al., 2007). The most significant property of dichloroacetate is that it has no effect on normal cells (Bonnet, et al., 2007). Our results corroborate our hypothesis that dichloroacetate would inhibit cell proliferation and are highly indicative of the veracity of DCA as a cancer-targeting drug. The 20mM DCA concentration seemed to be most effective. In protocol A, after 168 hrs in 20mM DCA, K562 showed little to no proliferation. Protocol B showed similar results. This, however, does little to show the effects of DCA on normal stem cells. Thus, we tested 10mM and 20mM concentrations of DCA on normal, noncancerous stem cells (Sample BM2) and found that the cell concentrations did not significantly change. As evidenced in Protocol A and Protocol B, these results suggest that DCA decreases cell proliferation in cancer cells as its concentration increase.
Cho, Liu 15 In the future, more tests on normal stem cells and cancer stem cells will need to be performed. Since this is an ongoing project, we will aim to establish a more solid foundation testing the efficacy of DCA by testing more on normal stem cells and creating assays that involve normal and cancerous stem cells in a single culture. Discussion In our analysis of the differences between cancerous and normal stem cells, we investigated the gene expression of the Wilm’s Tumor (WT1) gene and its implications in our experiment. The WT1 gene controls cell proliferation and is often present in the case of a tumor, because rapid cell proliferation is involved. In our investigation, the K562 treated with 20mM of DCA exhibited a low expression of the WT1 gene—about the same level of expression as a normal, non-cancerous stem cell sample. In contrast, a sample of K562 not treated with DCA exhibited a much higher expression of the WT1 gene, as evidenced in Figure 9. “the Wilms’ tumor gene WT1 plays an important role in cell proliferation and differentiation” (Kanato et al., 2005). This explains why K562, a leukemia cell line would have a high expression of this gene. Normal stem cells would not because, although they self-renew themselves, they do not proliferate constantly like cancer cells. The results that we achieved were similar to the experimentation conducted at the University of Alberta. DCA had the effect of eradicating cancer stem cells and inhibiting cell proliferation, as well as leaving noncancerous cells unaffected. According to this article, cancer cells make energy through glycolysis, instead of making use of the mitochondria for energy. Cancer cells prefer glycolysis, because this process still operates in the absence or lack of oxygen to the rapidly multiplying cells. The mitochondria operates apoptosis, thus turning off the mitochondria prevents apoptosis from occurring. DCA attacks the glycolysis process by inhibiting the use of pyruvate by glycolysis through activation of oxidative phosphorylation in the mitochondria. We found that as the concentration of dichloroacetate increases, the cell concentration of K562 decreases. This shows the effectiveness of DCA on cancer cells. In order to validate our findings, we tested on normal stem cells with 5, 10, and 20 mM of DCA and found that the differences in the concentrations of the controls and the experimental samples were insignificant. This further supports the DCA investigation at
Cho, Liu 16 the University of Alberta. Scientists researching this compound discovered that DCA inhibits cancer and does not harm normal cells. In order to mimic what occurs in the human body, tests of DCA in an environment with cancer and normal stem cells would be recommended. The current problem in chemotherapy of targeting cancer cells while leaving cancer stem cells viable can be solved in the future with more research involving DCA. Our findings and novel application of DCA on stem cells serve as a foundation on which to continue research in oncology.
Inquiry Process Jimmy Liu Investigation in the field of oncology was both challenging and rewarding for me. Although the rigor of IMSA’s advanced science classes prepared me well for our experimentation, there is no substitution for real labwork at an institution such as Children’s Memorial Hospital. Working at the Stem Cell Transplant Lab was a unique experience that challenged me to stretch the limits of my creative thinking and integrate novel methods of solving ageold problems. Our advisers provided invaluable guidance, but ultimately, because the project was our own, it was our duty to determine what tests to conduct and to discover new approaches to test our hypothesis. Instead of supplementary worksheets, we conducted all of our own background research and used creative problem solving in our calculations. This project has taught me that creative thinking is essential, especially in science. If our first approach didn’t work, we were forced to analyze and synthesize an explanation, and formulate a solution. For example, when we first diluted DCA with water, all of the cells died. After thinking back to our biology courses, we realized that water would cause the cells to become hypotonic and burst. Luckily, our problem solving, coupled with the advice of our advisers, allowed us to achieve our ultimate goal: learning through experimentation. An obvious task we wanted
Cho, Liu 17 to accomplish was to discover the effects of dichloroacetate on stem cells; however, we also set our mind to learning through our mistakes and experiencing real lab situations. Although more research and experimentation will be needed to confirm our findings, we succeeded in touching on the effects of DCA. Previous to starting our investigation, I had read about cancer stem cells and new implications in the cancer field. We started our project on a broad topic of the difference between normal and cancer stem cells, because we wanted to be in the forefront of cancer research. A couple of weeks into the SIR program, we decided to narrow our path of investigation to specifically looking at the effects of DCA on cancer and normal stem cells. DCA was found to inhibit cancer growth while leaving normal cells unaffected, so in our experiment we wanted to confirm this discovery. Idyllically, we wanted to test several samples; however, since we were receiving discarded blood samples, we had a difficult time attaining enough samples for stem cells. Many times we did not have enough cells to create a lasting sample and many times these samples were not fresh. We overcame this by replacing cancer stem cells with the K562 cell line. However, we did not have replacements for the normal bone marrow stem cells, so we used what was available for us. The whole experience was a learning process. At the beginning, we did not wash out the TrypsinEDTA. Due to this, the data we compiled were invalid. Even with this fallback, we advanced in our investigation and learned from our inaccuracies. Justina Throughout inquiry, I gained many valuable laboratory skills and built relationships that I never would have without the research opportunity. I discovered that the best way to learn is through experiences. IMSA’s advanced science classes prepared me for basic labwork; however, actually being in a lab and conducting high-level research challenged me to go beyond my limits. I was able to build upon my basic lab skills and accomplish
Cho, Liu 18 more than I previously expected. Ms. Marie Olszewski and Wei Huang, the lab technician, helped us a great deal throughout our project. Marie explained the basic procedures we would most likely be using, and then allowed us to conduct the experiments ourselves. Since this was an independent investigation separate from our mentors’, we learned old procedures and even created our own. The off-campus SIR experience was different than anything I have done in school, because I did my own background research, solved any problems by myself (without the help of a teacher), and did not have limits like school assignments. I felt a lot more independent being able to do my own experiments. I started the project by myself with little direction to what I wanted to accomplish. My current partner joined me in my investigation and brought with him his own knowledge of recent cancer research. He suggested testing dichloroacetate as another assay to assess stem cells. The general question, “What is the difference between cancer and normal stem cells?” evolved into, “What are the effects of dichloroacetate on cancer and normal stem cells with respect to cell concentration and gene expression?” Our mentor emphasized that experimenting was a learning process, and when we made mistakes she did not correct us, because she wanted us to learn by ourselves. In one instance we diluted the DCA for our experiment with water and later discovered that the cells had all died. We pondered over how the cells had died, and figured out that the cells became hypotonic and burst. Thus, we changed our procedure to diluting the DCA with
Cho, Liu 19 RPMI tissue culture instead of water. We also had trouble getting the results we wanted because our blood samples were not fresh. Since we were receiving discard samples, blood samples were not readily available. We overcame this by using K562, the human myeloid leukemia cell line, instead of cancer stem cells. This was a valid replacement because K562 has many similar characteristics to hematopoeitic cancer stem cells. However, we did not have a replacement for normal stem cells, so we had to use what was available to us. These drawbacks did not hinder our investigation. We pulled through and achieved many great results.
References (2007). Gene Almanac. Retrieved March 5, 2008, from Dolan DNA Learning Center Web site: http://www.dnalc.org/ddnalc/resources/pcr.html (2007). Graduate School of Biomedical Sciences. Retrieved March 5, 2008, from University of Medicine and Dentistry of New Jersey Web site: http://www.umdnj.edu/gsbsnweb/stemcell/scofthemonth/scofthemonth2/gut/1.jpg Alberts, B., (2002). Molecular Biology of the Cell. New York: Garland Science.
Appendix E: Stem Cell Markers (2001, June 17). Retrieved September 26, 2007, from http://stemcells.nih.gov/info/scireport/appendixe Bonnet, S., Archer, S. L., Allalunis-Turner, J., & Haromy, A.(2007). A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell, 11, 37-51.
Cho, Liu 20 Breems, D., Löwenberg, B. (2007). Acute Myeloid Leukemia and the Position of Autologous Stem Cell Transplantation. Semin Hematol.. 44, 259-266. Hernandez-Cabellero, E., Mayani, H., Monetsinos, J.J., Arenas, D., Salamanca, F., & Penaloza R.(2007). In vitro leukemic cell differentiation and WT1 gene expression. Leukemia Research. 31(3), 395-397. Hidehiko, A., Masato, W., Etsuko, T., Chikako, N., Itsuro, K., & Nobuhiko, E. (2006). WT1 tumor gene study using real-time quantitative polymerase chain reaction. Journal of Analytical Bio-Science. 29(3), 261-266. Jørgensen, H., Holyoake, T. (2007). Characterization of cancer stem cells in chronic myeloid leukaemia. Biochem Soc Trans.. 35, 1347-51. Kanato, K., Hosen N., Yanagihara, M., Nakagata, N., Shirakata, T., & Nakasawa T. (2005). The Wilms’ tumor gene WT1 is a common marker of progenitor cells in fetal liver. Biochemical and Biophysical Research Communications. 326 (4), 836843. Lee, J. T., & Herlyn, M. (2007). Old disease, new culprit: Tumor stem cells in cancer [Electronic version]. Journal of Cellular Physiology. 213(3), 603-609. from PubMed. Zou, G. (2007). Cancer stem cells in leukemia, recent advances. Journal of Cellular Physiology, 213(2), 440-444. Leukemia and Lymphoma Society. (2007, June). Leukemia Facts and Statistics. Leukemia, Lymphoma, Myeloma Facts 2007-2008. Retrieved September 26, 2007, from http://www.leukemia-lymphoma.org/all_page?item_id=9346& viewmode=print Morrison, S. J., & Kimble, J. (2006). Asymmetric and symmetric stem-cell divisions in development and cancer. Nature, 441, 1068-1074. National Center for Health Statistics. (2007, October 4). Fast Stats A to Z. Retrieved October 8, 2007, from http://0-www.cdc.gov.mill1.sjlibrary.org/
Cho, Liu 21 nchs/fastats/deaths.htm National Institutes of Health. (2001, June 17). Hematopoietic Stem Cells. Stem Cell Information. Retrieved September 16, 2007, from http://stemcells.nih.gov/ info/scireport/chapter5.asp Nicholas, W. (2006, February 21). Stem Cells May Be Key to Cancer. Retrieved September 12, 2007, from http://www.nytimes.com/2006/02/21/health/21canc.html?_r=1&oref=slogin Rajasekhar, V., & Dalerba, P.(2007). Stem Cells, Cancer, and Context Dependence. Stem Cells. [electronic version] Reya, T., Morrison, S. J., Clarke, M. F., & Weissman, I. L. (2001). Stem cells, cancer, and cancer stem cells [Electronic version]. Nature, 414, 105-111. Ru, Y., & Zhao, S. (2007). Ultrastructural Characteristics of Bone Marrow in Patients with Hematological Disease: A Study of 13 Cases.. Ultrastruct Pathol. 5, 327332. Sales, K., Winslet, M., & Seifalian A. (2007). Stem Cells and Cancer: An Overview. Stem Cell Rev. [electronic version] Young, N.S., & Hwang-Chen, S.P. (1981). Anti-K562 cell monoclonal antibodies recognize hematopoietic progenitors. Proc Natl Acad Sci U S A. 11, 7073-7077.
Cho, Liu 22
Appendix A. Flow-cytometry. Introduction: Flow-cytometry analysis was used in our experimentation, but ultimately did not support our conclusions or further our experiments or results. However, it did force us to question why the results we achieved in the flow-cytometry were not those that we expected. As a result, the flow-cytometry section of our experimentation caused us to further research certain stem cell markers and their applicability in our experimentation. The principles of flow-cytometry are relatively simple; light scattering and fluorescent tagging are employed in this procedure, and a laser is usually the light source. When the light from the laser strikes a particle of interest, it is excited to the next energy level, and a photon is released. Flow-cytometry is unique in that it measures fluorescence per cell. Our flow-cytometry results consisted of two-parameter histograms, with different
Cho, Liu 23 antibodies on the x- and y-axis. Each dot on the histogram represents a cell or particle; the histogram provides an excellent visual representation of the cells tagged by fluorescence. The following results were a part of our investigation, but did not add to our conclusion. The types of stem cells present in a culture can be determined with cell surface markers. The cell markers bind to specialized proteins on the surface of every cell in the body called receptors. Fluorescent tags are attached to surface markers (Figure 10) and these fluorescent tags are then detected via flow cytometry, a technique which picks up fluorescent light and thus identifies the cell surface markers.
Table 1. The types of cells that are positive for various cell surface markers and the sources used for this experiment. Cell Surface Marker CD34 (Miltenyi Biotec) CD123 (BD Pharmingen)
Cell Type Hematopoeitic Progenitor cells (stem cells) Multipotential hematopoietic stem/precursors. Expressed in cells that
CD133 (Miltenyi Biotec)
proliferate. Hematopoeitic stem cells (younger stem
CD14 (BD Simultest) CD45 (BD Simultest)
cell) Monocyte Lymphomas, Bcell chronic lymphocytic leukemia White cells, panleukocyte Fluorescent Tagging of Cells
Cho, Liu 24
Source: Appendix E: Stem Cell Markers (2001). Figure 10. Identifying Cell Surface Markers Using Fluorescent Tags. Methods and Materials: In order to determine the types of cells in our sample, we performed flow cytometry. Falcon tubes were labeled with the following monoclonals: • • • •
Isotype control (G1/G2a) CD14+/CD45+ CD133+/CD34+ CD123+/CD34+
Then to each of the test tubes, 10 microliters (uL) of the appropriate monoclonal antibody and 50 uL of cells were added. The tube is gently vortexed and then incubated for 10-15 minutes at room temperature away from light. We added 2 mL of 1xBD FACS Lyse working solution and then vortexed and incubated for a maximum of 10 minutes. This solution was centrifuged at 1800 rpm for 5 minutes. The supernatant was poured out and 2 mL of wash solution was added. After the second wash, we decanted the supernatant and added 350 uL of 1% Paraformaldehyde. Finally, we put the sample in the vortex and refrigerated it until it was ready to use. Through flow cytometry, certain characteristics of the normal and cancer cells were determined. In protocol a, 23.53% of the K562 control cells were double positive for CD123 and CD 34. CD123 is a cell surface marker for cells capable of proliferation, while CD34 is a marker for hematopoietic stem cells. The percentage of cells that are both positive steadily increases as the concentration increases. Table 2 shows the results
Cho, Liu 25 of flow cytometry detecting the markers of CD 123 and CD34 on the control K562, K562 treated with 10mM DCA, and K562 treated with 20 mM DCA. Table 2. The percentage of cells that are both CD123 and CD34. Flow cytometry was performed to determine the types of cell surface markers on the cells. Protocol a CD123+ and CD34+ K562 control 28.53% 10mM DCA treated 49.86% 20mM DCA treated 48.95% Flow cytometry was also performed on normal stem cells and only 1.50% of the cells were both CD123 and CD34 positive.
The Detection of Differences Between Normal and Cancerous Stem Cells Through Analysis of Morphology, Gene Expression, and Effects of Dichloroacetate Hyunjii Cho and Jimmy Liu, advisers: Marie Olzewski, and Dr. Morris Kletzel at the Children’s Memorial Hospital Stem Cell Laboratory Abstract Developments in oncology research point to stem cells as a promising, new means through which to cure cancer. We observed the morphology, gene expression, and response to dichloroacetate (DCA) of normal and cancerous stem cells. Mononuclear cells isolated from human blood were cultured for experimentation. We tested several different DCA concentrations to determine if cancer cells and normal cells die in DCA. To determine which stem cell markers exist on the cells, we performed flow-cytometry, a technique used to analyze physical and chemical characteristics of single cells flowing through an electronic detection apparatus. We also used RT-PCR to determine the genes expressed. When DCA was applied to the experimental group, the concentration of cells was successfully suppressed, implying that DCA has the effect of prohibiting cell proliferation. This implies the restoration of apoptosis in cancerous cells and the preservation of normal cells. DCA could be pursued as a cancer therapy because of its preferential effect on cancerous cells, and consequently, we plan to further test its potential. In particular, we plan to test DCA on cell cultures with both normal and cancerous cells combined; current tests have only tested DCA on normal and cancerous cells individually. Focusing Question What are the effects of dichloroacetate on normal and cancerous stem cells with respect to cell concentration and gene expression? Introduction We decided to research oncology because of its significance in today’s society. More specifically, we focused on the elucidation of the differences between cancerous and normal adult stem cells by analyzing key characteristics such as morphology with
Cho, Liu 2 stem cell markers and gene expression, because we firmly believe that it will be essential in discovering the mechanisms through which cancer progresses. Another goal of our experimentation was to test the efficacy of dichloroacetate (DCA) as a targeted cancer therapy. DCA is a promising compound in cancer research because of its nontoxicity and versatility. The mechanism through which it acts is unique: it exploits the dysfunctional mitochondria, a characteristic unique to cancer cells. Because of this, as well as its small size, it is able to treat various types of cancer, including brain, lung and breast cancers. DCA restores apoptosis in cancer cells while leaving normal cells unaffected. The applications of dichloroacetate as a novel cancer therapy are limitless, because DCA has been previously used to treat rare metabolic disorders; it is known to be relatively safe and non toxic. Our study further tests the compound in the context of the immortalized human myeloid leukemia cell line K562. Previous research has been conducted with DCA testing its effectiveness on normal and cancerous cells, but our experimentation adopts a novel approach through analyzing the effects of DCA on normal and cancerous stem cells (Bonnet, Archer, Allalunis-Turner, & Haromy, 2007). Hematopoietic stem cells are isolated from the peripheral blood or bone marrow and are capable of selfrenewal and differentiation into specialized cells. Apoptosis, programmed cell death, is necessary in order to prevent an excessive number of cells. Normal apoptotic functions are not present in cancerous stem cells, allowing unlimited cell regeneration. In healthy stem cell division, the cell regenerates itself and also creates a progenitor cell. Although the progenitor cell cannot regenerate itself, it is capable of dividing indefinitely into mature specialized cells (Lee & Herlyn, 2007). The progenitor cell then matures and become a specialized cell. Stem cells are characterized by both “selfrenewal and their ability to produce cells that differentiate” (Morrison & Kimble, 2006), called progenitor cells. Stem cells divide using two strategies: asymmetric and symmetric stemcell divisions. In
Cho, Liu 3 asymmetric stemcell division, stem cells divide to produce one identical daughter of itself, and one daughter that is capable of differentiating. Through this process, stem cells are unable to expand in number and thereby unable to produce stemcell pools that are needed for development and for cell regeneration after injury. Therefore, there must be another method that stem cells utilize to maintain control of their numbers. “Symmetric divisions are defined as the generation of daughter cells that are destined to acquire the same fate.” (Morrison et al., 2006). Through symmetric division, stem cells are able to divide into two stem cell daughter cells or two progenitor cells. Most stem cells are able to divide both asymmetrically and symmetrically, and these two modes are controlled by developmental and environmental signals. However, this balance is sometimes disrupted and defective in disease states.
Division of a Stem Cell
Source: University of Medicine and Dentistry at New Jersey (2007). Figure 1. Division of stem cells displaying selfrenewal and the progenitor cell. Stem cell matures into a progenitor cell, and differentiates into various other cells.
Cho, Liu 4 The K562 human leukemia cell line is strikingly similar to stem cells. According to Young and Hwangchen, K562 has characteristics of selfrenewal and pluripotency similar to those of stem cells (1981, p.7073). Comparable to leukemic stem cells, K562 also expresses the WT1 gene (Hidehiko, Masato, & Etsuko, 2006). According to Kanato, Hosen, and Yanagihara, WT1 expression is restricted to hematopoietic stem cells or progenitor cells. The Wilm’s Tumor 1 gene (WT1) is overexpressed in leukemia, thus explaining the proliferation of blood cells causing leukemia (2005). In normal subjects, there is low or undetectable expression of the WT1 gene; however, this gene is widely expressed in leukemic cell lines, as well as in the majority of lymphoid and myeloid leukemias of childhood and adulthood (HernandezCaballero, Mayani, & Montesinos, 2007). The rationale for testing the efficacy of DCA on stem cells is the methods of current chemotherapy and its success rate. Recent research in oncology points to stem cells as a novel method through which to study cancer. When a tumor is targeted, often there is a stem cell that differentiates into progenitors. The tumor is essentially composed of cancerous cells surrounding a few cancerous stem cells. The reason that many patients come out of remission is that although the cancer cells are eradicated, the cancerous stem cells remain viable (Lee & Herlyn, 2007). This poses a problem, as cancerous stem cells have unlimited potential to differentiate, thus causing cancer to relapse (Lee & Herlyn, 2007). A secondary method used to measure the efficacy of DCA is the analysis of the expression of the Wilm’s Tumor gene (WT1). The presence of this gene indicates tumor progression, which requires excessive cell proliferation (Kanato, Hosen, Yanagihara, Nakagata, Shirakata, & Nakasawa, 2005). If WT1 expression is not detected, then the culture of cells will not proliferate rapidly and is not cancerous.
Cho, Liu 5 In a previous study at the University of Alberta, it was discovered that DCA normalizes a dysfunctional mitochondria, thus restoring apoptotic function (Bonnet et al., 2007). More specifically, cancer is unique in that it has a high membrane potential, downregulated K+ channels, and metabolizes through glycolysis. Membrane potential is the difference in electrical voltage between the inside and outside of the cell membrane (Alberts, 2002). Regular membrane potential allows for a stable cell environment; DCA was recently discovered to decrease the high mitochondria membrane potential. In addition, DCA upregulates the K+ channels and shifts metabolic activity from the cytoplasmbased glycolysis to the mitochondriabased glucose oxidation (Bonnet et al., 2007). By testing the efficacy of DCA on stem cells, we hope to further confirm the efficacy of DCA. We hypothesized that DCA would have a positive effect in restoring normal apoptosis, or programmed cell death, in cancer stem cells, and effectively inhibit cell proliferation. We also hypothesized that DCA would leave normal stem cells unaffected. Materials and Methods: In order to proceed with the experiment, blood samples that were ready to be discarded from the Children’s Memorial Hospital were obtained. K562 cells were used in place of cancer stem cells due to their similarities: K562 cells, like cancer stem cells, are able to self-renew and proliferate, as well as express the Wilm’s Tumor 1 (WT1) gene, which is exhibited in stem cells. Mononuclear cells, or stem cells were isolated by creating a density gradient. Before diluting the blood, an initial cell count was taken with the Cell Dyn 1700 machine. We then created a density gradient with the use of FicollPaque, underlayering blood with Ficoll-Paque. To keep the sample sterile, we avoided contact between the Ficoll-Paque bottle and the test tube when adding the Ficoll-Paque. Blood diluted with phosphate buffered saline (PBS) in a ratio of 1:1 and was added to this test tube. The test tube was balanced in a centrifuge and spun at 3000 rpm for 10 minutes
Cho, Liu 6 without a brake. Mononuclear cells were removed from the resulting contents, as shown in Figure 2.
Figure 2. Density gradient using Ficoll-paque.
These cells were then collected and washed with PBS and spun in the centrifuge for 5 minutes at 3000 rpm. Afterwards, the PBS and plasma were poured out and the compacted cell pellet remained at the bottom (Figure 3).
Cho, Liu 7
Figure 3. Pellet at the bottom of a test tube formed from the accumulation of mononuclear cells.
Approximately 2 mL of RPMI tissue culture was added in preparation of culturing the cells. The cell pellet was resuspended and mixed well. A final cell count was taken with the Cell Dyn 1700 in order to calculate the amount of cells per well would be needed. In our experiment, we used about 1.0x106-2.0x106 cells per well for normal bone marrow stem cells, while we used about 0.1 x 106 cells for K562, since this human immortalized myelogenous leukemia cell line proliferates rapidly. In order to culture these cells, 1mL of RPMI was added to each well and the plate was placed in the incubator. The RPMI was changed roughly every week for normal bone marrow stem cells, and about every 3 days for the K562 cell cultures. Cell viability was tested with Trypan Blue. 10 µL of the cell sample was mixed with 20 µL of Trypan Blue. Then this mixture was inserted into a sterilized hemacytometer; the hemacytometer was sterilized to remove any residue before examining the sample of cells and to avoid contamination. The cells were viewed under a microscope and viable and dead cells were counted. This method works because when a cell is not viable, the function of its semi-permeable membrane is compromised. This allows the penetration of Trypan Blue into the cell; unviable cells are permeated with
Cho, Liu 8 Trypan Blue, while the semi-permeable membrane of living cells prevents the penetration of Trypan Blue; the cell is not blue. For our DCA experiment, we made 5, 10, 20, and 40 mM concentrations of DCA in RPMI solution. To the 1 mL solution of cell culture we added 1 mL of the DCA solution. Mixing the DCA solution to another 1 mL solution diluted the concentration of DCA by half. Thus actual 5, 10, 20, and 40 mM concentrations of DCA became 2.5, 5, 10, and 20 mM concentrations, respectively. DCA was added to the cells after a few days of culturing them. Cell concentrations of the cells in DCA were measured after 48 hours for normal stem cells, and 96 and 168 hrs for K562 cells. We also used reverse transcriptase-PCR to analyze the effect of DCA on the K562. Total RNA was extracted using QIAamp RNA Blood mini-kit (QIAGEN, Inc., Valencia, CA, USA). A 2-step reverse transcriptase (RT)-PCR in a 20-uL reaction volume with 1 microgram (ug) of total RNA from each sample was conducted. The reaction buffer included 4 uL of 25 nM MgCl2, 8 uL of dNTPs, 1 uL of Oligo d(T)16, 2 uL of 10xPCR buffer II, 1 uL of RNase inhibitor, and 1 uL of Moloney Murine Leukemia Virus RT (Applie Biosystem, Foster City, CA, USA). We used primer pairs that were designed to locate particular nucleotide fragments for mRNA of the WT1 gene. Reverse transcription was incubated at 42 degrees Celsius in a water bath for 60 minutes. The it was denatured at 96 degrees Celsius in a thermal cycler for 10 minutes. 10 microliters of the complementary DNA was amplified during the 1st round of PCR. There was an initial 30 cycles of denaturation for 5 minutes, 5 seconds at 95 degrees Celsius amplification, annealing at 55 degrees Celsius fir 5 seconds, and extension at 72 degrees Celsius for 10 seconds. A second round was performed by taking 1 uL of the 1st round of amplified product and reamplifying with inner primers utilizing the LightCycler System with SYBR-Green 1 RNA master mix reagent (Roche Diagnostics, Biochemica, Indianapolis, IN, USA). Once PCR was complete, the LightCycler Software calculated the concentration of target molecules. Using a 10-fold serial dilution of K562 leukemia cell line and the Bone Marrow 2 (BM2) sample, a standard curve was generated for each sample. Values of 1x 100ng/uL and higher were considered WT1 positive. PCR products
Cho, Liu 9 were then run through a 1.5% agarose gel electrophoresis. Finally the data was analyzed in the form of graphs.
Results Figure 4 displays the increase in K562 cell concentration as time increases, thus confirming the indefinite proliferation of K562. The initial cell concentrations of K562 at Day 0 were 0.1 thousand per microliter (K/uL). K562 Control (Jan. 16.08- Jan. 30. 08) 3.5
1.0877x
y = 0.0385e 2 R = 0.9541
3
Concentration (K/uL)
2.5
2
1.5
1
0.5
0 D0
D3
D10
D14
Time (days)
Figure 4. Bar graph of the cell concentration as time increases. K562 was cultured and the cell concentration of the controls (without DCA) were recorded at various times in a 14 day period. The graph in figure 5 displays a decrease in cell concentration of K562 as the DCA concentration increases.
Cho, Liu 10
Figure 5. Protocol a: The effects of increasing DCA concentration on the cell concentration of K562. Cell concentration (K/uL) of K562 cell cultures with no DCA and cultures with 2.5 mM, 5mM, 10mM, and 20mM were observed after 168 hours after the addition of DCA. The highest concetrations of DCA from protocol a were tested again in another set of experiments, protocol b. The resulting K562 cell concentrations in 10 mM and 20mM DCA after 96 hours of the addition of DCA shows to be far less than that of the control measured at the same time.
Cho, Liu 11 Figure 6. Protocol b: K562 cell concentrations on day 14 of the culture and 96 hours after adding DCA to certain cultures. 10mM and 20mM DCA concentrations were tested and compared to the day 14 control culture. Protocol c consisted of Bone Marrow Sample #2 (BM2) normal bone marrow stem cell cultures at different concentrations. The effects of the various DCA concentrations on BM2 were relatively similar. At day 0 of the culture, the initial cell concentration was 2.0K/uL. At day 16 of the culture and 48 hours after the addition of DCA to some of the wells of the culture, the sample was observed. The concentrations were relatively around 1.7K/uL. There were no significant differences between the BM2 cell concentration of the control and the DCA-treated cultures. Protocol c: BM2 March 7 Day 16 (48Hrs in DCA) 2 1.8 1.6
1.72
1.7
Control
5mM
1.72
1.7333333
10mM
20mM
Cell Concentration (K/uL)
1.4 1.2 1 0.8 0.6 0.4 0.2 0 DCA Concentration (mM)
Figure 7. Protocol c: BM2, a normal stem cell sample, was treated with various concentrations of DCA. The resulting cell concentrations after 48 hrs of immersion in DCA were compared to the control, which had been cultured for a total of 14 days. A secondary result of experimentation is the analysis of gene expression. Again, the most significant difference can be evidenced through a comparison of non-DCA treated cultures and DCA-treated cultures. As shown in Figure 8 and Figure 9, when a pristine culture of K562 cells is used as the baseline, the two cultures treated with 20 mM of DCA from both protocol a and b have low expressions of WT1, the cell proliferation gene. BM2, the normal stem cell sample, also has a very low, almost undetectable, expression of WT1. The control K562 samples and the 10mM DCA concentration K562
Cho, Liu 12 samples all express the WT1 relatively higher than the 20mM DCA treated K562 samples.
Figure 8. WT1 expression in K562 controls, 10mM and 20mM DCA treated K562 samples, and BM2. The resulting data was examined through the LightCycler Data Analysis.
Figure 9. WT1 expression show in bar graphs for K562 controls, 10mM and 20mM DCA treated K562 samples, and BM2.
Conclusion
Cho, Liu 13 In the first set of experiments (protocol A), various concentrations of DCA were tested on cultures of 2x106 cells per well: K562 control group (n=2), K562 group with 2.5mM (n=2), 5mM DCA (n=2), K562 group with 10mM DCA (n=2). The control group, which was not treated with DCA, continued normal cell proliferation. The DCA-treated groups exhibited a decreased concentration of cells, indicating decreased cell proliferation. Using regression analysis, we determined that the concentration of cells decreased exponentially as the concentration of DCA increased. The second set of experiments (protocol B) consisted of testing 10mM and 20mM DCA concentrations on K562. 10mM and 20mM DCA concentrations were chosen for the second set of experiments (protocol B) on K562 because they exhibited almost no increase in cell concentration. The initial cell concentration was 0.1 K/uL, while the cell concentrations of K562 cells in 10mM and 20mM DCA for 168 hrs only had 0.2 K/uL cell concentrations. This is not a significant increase and can be accounted for by few trials, and human and machine (Cell Dyn 1700) error. Protocol B confirmed that DCA may inhibit cell proliferation because it also showed that cell concentrations did not increase significantly from the initial cell concentration of 0.1 K/uL and they also did not proliferate as much as the control did. RT-PCR was performed on protocol A and protocol B. The expression of WT1 in protocol A and B was measured through RT-PCR. In protocol A, the K562 group treated with 10mM of DCA showed that the cells still expressed the Wilm’s tumor suppressor gene (WT1), which regulates cell proliferation. However, the cells in protocol A that were treated with 20mM of DCA did not significantly exhibit the WT1 gene. The test was repeated for protocol B and similar results were achieved. These results indicate that a 20mM concentration of DCA is more effective than a 10mM concentration because it successfully inhibits the expression of WT1. They also support the correlation between increased DCA concentration and decreased cell concentration. The effects of DCA were also tested on normal, non-cancerous adult stem cells (Sample BM2). DCA had no significant effect on the cell concentrations of BM2. This shows that while DCA affects cancerous cells, it does not affect normal stem cells. More
Cho, Liu 14 tests will need to be performed on normal cells in the future, in order to wholly support our conclusion. Due to the lack of normal bone marrow stem cells available, we were not able to perform many tests on normal stem cells. Our findings in the morphology through analysis of stem cell markers contradict the discoveries in WT1 expression, and were thus, unexpected. Through flow cytometry, we found that the percentage of K562 cells that are both CD123 and CD 34 positive increases as the concentration of DCA increases. This means that the percentage of K562 cells that are capable of proliferating have a positive correlation with DCA concentrations. The stem cell marker CD123 may be present on cells for more than just capability of proliferation; therefore, the RT-PCR results would prove to be more reliable. Gene expression is more specific in determining function than the stem cell markers. In order to understand why the two assays contradict each other, further research will be conducted attempting to determine the cause of the presence of CD123 on cells. One might hypothesize that the CD123 stem cell marker has other functions than indicating cell proliferation. The prospect of dichloroacetate as a novel, therapeutic cancer treatment is realistic because it restores mitochondrial function, one of the fundamental pathways unique to the progression of cancer. Additionally, as it has been used for many decades in the treatment of metabolic diseases, it is known to be relatively non-toxic (Bonnet, et al., 2007). The most significant property of dichloroacetate is that it has no effect on normal cells (Bonnet, et al., 2007). Our results corroborate our hypothesis that dichloroacetate would inhibit cell proliferation and are highly indicative of the veracity of DCA as a cancer-targeting drug. The 20mM DCA concentration seemed to be most effective. In protocol A, after 168 hrs in 20mM DCA, K562 showed little to no proliferation. Protocol B showed similar results. This, however, does little to show the effects of DCA on normal stem cells. Thus, we tested 10mM and 20mM concentrations of DCA on normal, noncancerous stem cells (Sample BM2) and found that the cell concentrations did not significantly change. As evidenced in Protocol A and Protocol B, these results suggest that DCA decreases cell proliferation in cancer cells as its concentration increase.
Cho, Liu 15 In the future, more tests on normal stem cells and cancer stem cells will need to be performed. Since this is an ongoing project, we will aim to establish a more solid foundation testing the efficacy of DCA by testing more on normal stem cells and creating assays that involve normal and cancerous stem cells in a single culture. Discussion In our analysis of the differences between cancerous and normal stem cells, we investigated the gene expression of the Wilm’s Tumor (WT1) gene and its implications in our experiment. The WT1 gene controls cell proliferation and is often present in the case of a tumor, because rapid cell proliferation is involved. In our investigation, the K562 treated with 20mM of DCA exhibited a low expression of the WT1 gene—about the same level of expression as a normal, non-cancerous stem cell sample. In contrast, a sample of K562 not treated with DCA exhibited a much higher expression of the WT1 gene, as evidenced in Figure 9. “the Wilms’ tumor gene WT1 plays an important role in cell proliferation and differentiation” (Kanato et al., 2005). This explains why K562, a leukemia cell line would have a high expression of this gene. Normal stem cells would not because, although they self-renew themselves, they do not proliferate constantly like cancer cells. The results that we achieved were similar to the experimentation conducted at the University of Alberta. DCA had the effect of eradicating cancer stem cells and inhibiting cell proliferation, as well as leaving noncancerous cells unaffected. According to this article, cancer cells make energy through glycolysis, instead of making use of the mitochondria for energy. Cancer cells prefer glycolysis, because this process still operates in the absence or lack of oxygen to the rapidly multiplying cells. The mitochondria operates apoptosis, thus turning off the mitochondria prevents apoptosis from occurring. DCA attacks the glycolysis process by inhibiting the use of pyruvate by glycolysis through activation of oxidative phosphorylation in the mitochondria. We found that as the concentration of dichloroacetate increases, the cell concentration of K562 decreases. This shows the effectiveness of DCA on cancer cells. In order to validate our findings, we tested on normal stem cells with 5, 10, and 20 mM of DCA and found that the differences in the concentrations of the controls and the experimental samples were insignificant. This further supports the DCA investigation at
Cho, Liu 16 the University of Alberta. Scientists researching this compound discovered that DCA inhibits cancer and does not harm normal cells. In order to mimic what occurs in the human body, tests of DCA in an environment with cancer and normal stem cells would be recommended. The current problem in chemotherapy of targeting cancer cells while leaving cancer stem cells viable can be solved in the future with more research involving DCA. Our findings and novel application of DCA on stem cells serve as a foundation on which to continue research in oncology.
Inquiry Process Jimmy Liu Investigation in the field of oncology was both challenging and rewarding for me. Although the rigor of IMSA’s advanced science classes prepared me well for our experimentation, there is no substitution for real labwork at an institution such as Children’s Memorial Hospital. Working at the Stem Cell Transplant Lab was a unique experience that challenged me to stretch the limits of my creative thinking and integrate novel methods of solving ageold problems. Our advisers provided invaluable guidance, but ultimately, because the project was our own, it was our duty to determine what tests to conduct and to discover new approaches to test our hypothesis. Instead of supplementary worksheets, we conducted all of our own background research and used creative problem solving in our calculations. This project has taught me that creative thinking is essential, especially in science. If our first approach didn’t work, we were forced to analyze and synthesize an explanation, and formulate a solution. For example, when we first diluted DCA with water, all of the cells died. After thinking back to our biology courses, we realized that water would cause the cells to become hypotonic and burst. Luckily, our problem solving, coupled with the advice of our advisers, allowed us to achieve our ultimate goal: learning through experimentation. An obvious task we wanted
Cho, Liu 17 to accomplish was to discover the effects of dichloroacetate on stem cells; however, we also set our mind to learning through our mistakes and experiencing real lab situations. Although more research and experimentation will be needed to confirm our findings, we succeeded in touching on the effects of DCA. Previous to starting our investigation, I had read about cancer stem cells and new implications in the cancer field. We started our project on a broad topic of the difference between normal and cancer stem cells, because we wanted to be in the forefront of cancer research. A couple of weeks into the SIR program, we decided to narrow our path of investigation to specifically looking at the effects of DCA on cancer and normal stem cells. DCA was found to inhibit cancer growth while leaving normal cells unaffected, so in our experiment we wanted to confirm this discovery. Idyllically, we wanted to test several samples; however, since we were receiving discarded blood samples, we had a difficult time attaining enough samples for stem cells. Many times we did not have enough cells to create a lasting sample and many times these samples were not fresh. We overcame this by replacing cancer stem cells with the K562 cell line. However, we did not have replacements for the normal bone marrow stem cells, so we used what was available for us. The whole experience was a learning process. At the beginning, we did not wash out the TrypsinEDTA. Due to this, the data we compiled were invalid. Even with this fallback, we advanced in our investigation and learned from our inaccuracies. Justina Throughout inquiry, I gained many valuable laboratory skills and built relationships that I never would have without the research opportunity. I discovered that the best way to learn is through experiences. IMSA’s advanced science classes prepared me for basic labwork; however, actually being in a lab and conducting high-level research challenged me to go beyond my limits. I was able to build upon my basic lab skills and accomplish
Cho, Liu 18 more than I previously expected. Ms. Marie Olszewski and Wei Huang, the lab technician, helped us a great deal throughout our project. Marie explained the basic procedures we would most likely be using, and then allowed us to conduct the experiments ourselves. Since this was an independent investigation separate from our mentors’, we learned old procedures and even created our own. The off-campus SIR experience was different than anything I have done in school, because I did my own background research, solved any problems by myself (without the help of a teacher), and did not have limits like school assignments. I felt a lot more independent being able to do my own experiments. I started the project by myself with little direction to what I wanted to accomplish. My current partner joined me in my investigation and brought with him his own knowledge of recent cancer research. He suggested testing dichloroacetate as another assay to assess stem cells. The general question, “What is the difference between cancer and normal stem cells?” evolved into, “What are the effects of dichloroacetate on cancer and normal stem cells with respect to cell concentration and gene expression?” Our mentor emphasized that experimenting was a learning process, and when we made mistakes she did not correct us, because she wanted us to learn by ourselves. In one instance we diluted the DCA for our experiment with water and later discovered that the cells had all died. We pondered over how the cells had died, and figured out that the cells became hypotonic and burst. Thus, we changed our procedure to diluting the DCA with
Cho, Liu 19 RPMI tissue culture instead of water. We also had trouble getting the results we wanted because our blood samples were not fresh. Since we were receiving discard samples, blood samples were not readily available. We overcame this by using K562, the human myeloid leukemia cell line, instead of cancer stem cells. This was a valid replacement because K562 has many similar characteristics to hematopoeitic cancer stem cells. However, we did not have a replacement for normal stem cells, so we had to use what was available to us. These drawbacks did not hinder our investigation. We pulled through and achieved many great results.
References (2007). Gene Almanac. Retrieved March 5, 2008, from Dolan DNA Learning Center Web site: http://www.dnalc.org/ddnalc/resources/pcr.html (2007). Graduate School of Biomedical Sciences. Retrieved March 5, 2008, from University of Medicine and Dentistry of New Jersey Web site: http://www.umdnj.edu/gsbsnweb/stemcell/scofthemonth/scofthemonth2/gut/1.jpg Alberts, B., (2002). Molecular Biology of the Cell. New York: Garland Science.
Appendix E: Stem Cell Markers (2001, June 17). Retrieved September 26, 2007, from http://stemcells.nih.gov/info/scireport/appendixe Bonnet, S., Archer, S. L., Allalunis-Turner, J., & Haromy, A.(2007). A mitochondria-K+ channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell, 11, 37-51.
Cho, Liu 20 Breems, D., Löwenberg, B. (2007). Acute Myeloid Leukemia and the Position of Autologous Stem Cell Transplantation. Semin Hematol.. 44, 259-266. Hernandez-Cabellero, E., Mayani, H., Monetsinos, J.J., Arenas, D., Salamanca, F., & Penaloza R.(2007). In vitro leukemic cell differentiation and WT1 gene expression. Leukemia Research. 31(3), 395-397. Hidehiko, A., Masato, W., Etsuko, T., Chikako, N., Itsuro, K., & Nobuhiko, E. (2006). WT1 tumor gene study using real-time quantitative polymerase chain reaction. Journal of Analytical Bio-Science. 29(3), 261-266. Jørgensen, H., Holyoake, T. (2007). Characterization of cancer stem cells in chronic myeloid leukaemia. Biochem Soc Trans.. 35, 1347-51. Kanato, K., Hosen N., Yanagihara, M., Nakagata, N., Shirakata, T., & Nakasawa T. (2005). The Wilms’ tumor gene WT1 is a common marker of progenitor cells in fetal liver. Biochemical and Biophysical Research Communications. 326 (4), 836843. Lee, J. T., & Herlyn, M. (2007). Old disease, new culprit: Tumor stem cells in cancer [Electronic version]. Journal of Cellular Physiology. 213(3), 603-609. from PubMed. Zou, G. (2007). Cancer stem cells in leukemia, recent advances. Journal of Cellular Physiology, 213(2), 440-444. Leukemia and Lymphoma Society. (2007, June). Leukemia Facts and Statistics. Leukemia, Lymphoma, Myeloma Facts 2007-2008. Retrieved September 26, 2007, from http://www.leukemia-lymphoma.org/all_page?item_id=9346& viewmode=print Morrison, S. J., & Kimble, J. (2006). Asymmetric and symmetric stem-cell divisions in development and cancer. Nature, 441, 1068-1074. National Center for Health Statistics. (2007, October 4). Fast Stats A to Z. Retrieved October 8, 2007, from http://0-www.cdc.gov.mill1.sjlibrary.org/
Cho, Liu 21 nchs/fastats/deaths.htm National Institutes of Health. (2001, June 17). Hematopoietic Stem Cells. Stem Cell Information. Retrieved September 16, 2007, from http://stemcells.nih.gov/ info/scireport/chapter5.asp Nicholas, W. (2006, February 21). Stem Cells May Be Key to Cancer. Retrieved September 12, 2007, from http://www.nytimes.com/2006/02/21/health/21canc.html?_r=1&oref=slogin Rajasekhar, V., & Dalerba, P.(2007). Stem Cells, Cancer, and Context Dependence. Stem Cells. [electronic version] Reya, T., Morrison, S. J., Clarke, M. F., & Weissman, I. L. (2001). Stem cells, cancer, and cancer stem cells [Electronic version]. Nature, 414, 105-111. Ru, Y., & Zhao, S. (2007). Ultrastructural Characteristics of Bone Marrow in Patients with Hematological Disease: A Study of 13 Cases.. Ultrastruct Pathol. 5, 327332. Sales, K., Winslet, M., & Seifalian A. (2007). Stem Cells and Cancer: An Overview. Stem Cell Rev. [electronic version] Young, N.S., & Hwang-Chen, S.P. (1981). Anti-K562 cell monoclonal antibodies recognize hematopoietic progenitors. Proc Natl Acad Sci U S A. 11, 7073-7077.
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Appendix A. Flow-cytometry. Introduction: Flow-cytometry analysis was used in our experimentation, but ultimately did not support our conclusions or further our experiments or results. However, it did force us to question why the results we achieved in the flow-cytometry were not those that we expected. As a result, the flow-cytometry section of our experimentation caused us to further research certain stem cell markers and their applicability in our experimentation. The principles of flow-cytometry are relatively simple; light scattering and fluorescent tagging are employed in this procedure, and a laser is usually the light source. When the light from the laser strikes a particle of interest, it is excited to the next energy level, and a photon is released. Flow-cytometry is unique in that it measures fluorescence per cell. Our flow-cytometry results consisted of two-parameter histograms, with different
Cho, Liu 23 antibodies on the x- and y-axis. Each dot on the histogram represents a cell or particle; the histogram provides an excellent visual representation of the cells tagged by fluorescence. The following results were a part of our investigation, but did not add to our conclusion. The types of stem cells present in a culture can be determined with cell surface markers. The cell markers bind to specialized proteins on the surface of every cell in the body called receptors. Fluorescent tags are attached to surface markers (Figure 10) and these fluorescent tags are then detected via flow cytometry, a technique which picks up fluorescent light and thus identifies the cell surface markers.
Table 1. The types of cells that are positive for various cell surface markers and the sources used for this experiment. Cell Surface Marker CD34 (Miltenyi Biotec) CD123 (BD Pharmingen)
Cell Type Hematopoeitic Progenitor cells (stem cells) Multipotential hematopoietic stem/precursors. Expressed in cells that
CD133 (Miltenyi Biotec)
proliferate. Hematopoeitic stem cells (younger stem
CD14 (BD Simultest) CD45 (BD Simultest)
cell) Monocyte Lymphomas, Bcell chronic lymphocytic leukemia White cells, panleukocyte Fluorescent Tagging of Cells
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Source: Appendix E: Stem Cell Markers (2001). Figure 10. Identifying Cell Surface Markers Using Fluorescent Tags. Methods and Materials: In order to determine the types of cells in our sample, we performed flow cytometry. Falcon tubes were labeled with the following monoclonals: • • • •
Isotype control (G1/G2a) CD14+/CD45+ CD133+/CD34+ CD123+/CD34+
Then to each of the test tubes, 10 microliters (uL) of the appropriate monoclonal antibody and 50 uL of cells were added. The tube is gently vortexed and then incubated for 10-15 minutes at room temperature away from light. We added 2 mL of 1xBD FACS Lyse working solution and then vortexed and incubated for a maximum of 10 minutes. This solution was centrifuged at 1800 rpm for 5 minutes. The supernatant was poured out and 2 mL of wash solution was added. After the second wash, we decanted the supernatant and added 350 uL of 1% Paraformaldehyde. Finally, we put the sample in the vortex and refrigerated it until it was ready to use. Through flow cytometry, certain characteristics of the normal and cancer cells were determined. In protocol a, 23.53% of the K562 control cells were double positive for CD123 and CD 34. CD123 is a cell surface marker for cells capable of proliferation, while CD34 is a marker for hematopoietic stem cells. The percentage of cells that are both positive steadily increases as the concentration increases. Table 2 shows the results
Cho, Liu 25 of flow cytometry detecting the markers of CD 123 and CD34 on the control K562, K562 treated with 10mM DCA, and K562 treated with 20 mM DCA. Table 2. The percentage of cells that are both CD123 and CD34. Flow cytometry was performed to determine the types of cell surface markers on the cells. Protocol a CD123+ and CD34+ K562 control 28.53% 10mM DCA treated 49.86% 20mM DCA treated 48.95% Flow cytometry was also performed on normal stem cells and only 1.50% of the cells were both CD123 and CD34 positive.
