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Linking Cataracts to Cancer: The Diagnosis of a Novel Oncoprotein to Predict Premature Basal-Like Breast Cancer Gregory Konar Massachusetts Academy of Mathematics and Science Abstract Alpha-basic crystallin and its associated gene CRYAB were studied for potential oncogenic activity in MDA-MB-231 basal like breast cancer cells under conditions of elevated stress with the inclusion of Rhodiola crenulata root extract. It was hypothesized that the protein will exhibit oncogenic activity in the stressed breast cancer cells and not in the normal immortalized cell line control, and the Rhodiola crenulata extract will suppress the protein. A Western Blot Assay was used for the testing with SDS-PAGE gel electrophoresis and protein exposure using chemoluminescense caused by Luminal. The proteins were lysed from the cell lines, and then they were separated and transferred onto a gel blot using the electrophoresis chamber. The proteins were blocked using the αb-crystallin antibody conjugated to HRP, and they were exposed using a GBox Exposure Machine. The protein was shown to be specific to only the breast cancer cells, and it had a 390% higher concentration in the MDA-MB-231 cells than in the normal cells. Rhodiola crenulata root extract showed marginal suppressive abilities, with the proteins being suppressed approximately 24% in both cell lines. The elevated expression of the alpha-basic crystallin in basal like breast cancer cells but not in normal breast cells proved that the protein is a feasible target for oncogenic activity in malignant diseases such as cancer, and that it should be looked at with association to poor clinical outcomes in the medical society. Introduction The primary objective of a significant amount of scientific research performed today is to discover a method to cure cancer; however, nobody has succeeded in fully curing it yet. An important part of the process of eliminating cancer cells and increasing survival chances is via early detection. Recent studies have produced increasingly positive results in accurately detecting premature cancer through protein expression. Proteins are ubiquitous molecules that are used in the function of both normal and malignant cells. In cancer cells, the genes that make up these proteins, referred to as oncogenes, play the greatest role in cell division through a process called the cell cycle. The cell cycle comprises four primary phases, which include S phase, M phase, G1 phase, and G2 phase. In a normal instance, the proteins would ensure that the DNA of the cell is copied during M phase, and that two daughter cells are produced during S phase. But when carcinogenic cells perform cellular division, mutated oncogenes target the growth regulators in the cell at G1 phase, and this causes the cell to stay in the division cycle and produce multitudes of cells in a shorter period of time than normal (Sherr, 1996). Because these oncogenes are found only in newly mutated carcinogenic cells, they have become a primary target in research on how cancer proliferates and metastasizes inside of the body. Targeting these anti-apoptotic or proliferator enhancers is achieved in multiple different ways, which range from examining active receptor kinases and growth factor regulators to detecting the expression of the oncoproteins associated with the oncogene. If scientists are able to detect all of the oncogenes and oncoproteins Linking Cataracts to Cancer 1 associated with the different types of cancer, then they will learn to predict premature cancer through simply observing the proteins and genes in an individual’s body. The protein that is the primary focus of this project is referred to as alpha-basic crystallin (αb- crystallin). Αb- crystallin is part of the crystallin protein family, and even though crystallin proteins are ubiquitous, αb- crystallin is found mostly in the eye lens or in some tissues throughout the body. Detection of small heat shock type proteins like αb- crystallin can be achieved through a variety of different methods, but for this testing the process that will be used is the Western Blot Assay. This means that the protein concentrations of the cell lines need to be found, and then gel electrophoresis needs to be performed in order to expose the proteins. The gel will then be transferred to a PVDF membrane and exposed to an antibody for 30 minutes, and then exposures will be taken of the proteins using an exposure machine. The exposures of this transfer blot should be analyzed at the 20 kilo-Dalton range in order to locate and detect the protein accurately. The phosphorylation status of the protein will also play a huge role in protein detection and activation. Phosphorylated proteins have a phosphate group added to certain amino acid locations found in the molecule. This additional phosphate group can either turn the protein on or off, which controls whether it will be expressed when exposed to its conjugate antibody. The amino acids that are associated with phosphorylation are serine, threonine, and tyrosine; in this testing the αb-crystallin protein will be phosphorylated at serine-59. Literature Review Basal Type Carcinoma of the Breast Ongoing concern in the area of breast cancer has led to not only increased awareness of this malignancy, but also to more research that focuses on different types of breast cancer. Cancer comprises four main groups of malignancies, carcinoma, sarcoma, lymphoma, and leukemia. All four of those groups affect different parts of the body, whether it is the blood, skin, or organs of an individual. Carcinoma is the most common form of cancerous malignancy that occurs in the body; it occurs when tissue cells become mutated and begin to form tumor cells. Common forms of carcinoma include most lung cancers, basal cell carcinomas, adenocarcinomas, and squamous cell carcinomas (Berman, 2011). Sarcomas are very rare strains of cancer; they form when cells that originate from bone, cartilage, muscle, or fat mutate and form malignant tumors. Sarcomas are the most difficult type of cancer to detect early because most patients feel little to no pain or discomfort in the afflicted area(s) (Bueckler, 2005). Lymphoma is also known as cancer of the lymphocytes, which are cells that work to form the immune system of an organism. Lymphoma, like almost all cancers, is considered to be an incurable disease, meaning that even the most aggressive forms of chemotherapy or radiation therapy will only alleviate the symptoms and not rid the body completely of the cancer. Leukemia is cancer of the blood or bone marrow, but is not just limited to cancerous malignancies. It covers an entire group of diseases that affect the blood, bone marrow, or lymphoid system referred to as hematological neoplasms. The biomarker that doctors use to determine whether leukemia is present in the body is by looking at the white blood cell count in an organism. If the number of cells is elevated greatly, then that signals that a malignancy is present. Basal cell carcinoma is classified under the carcinoma subtype and is the most common form of skin cancer in the United States. Over 2.8 million people in the United States are Linking Cataracts to Cancer 2 diagnosed with it every year. Basal cell carcinoma isn’t a highly fatal cancer with approximately 70,000 deaths occurring each year. It is a very slow growing cancer and can be highly mutilating if not treated immediately. Basal cell carcinoma is a nonmelanoma type of skin cancer, meaning that it is induced by overexposure to sunlight or x-rays; often this overexposure is caused by tanning beds or sun lamps (“Melanoma/Skin Cancer”, 2010). There are three main types of basal cell carcinoma, all of which are characterized by the lack of gene expression in a specific area, hormone receptor (HER2), estrogen receptor (ER), and progesterone receptor (PR). The people who are at the greatest risk of developing basal cell carcinoma are those with relatives who have had the disease, those who receive a heightened amount of sun exposure either from work or from tanning, and those who have many moles (because the cancer is apt to start growing from that area). The symptoms of basal cell carcinoma are subtle, painless, and usually resemble sores on the surface of the skin (Figure 1) that are discolored and will not heal. Figure 1. Morphology and cross section of basal cell carcinoma. The carcinoma at first looks like a mole or a sore, but it will not heal over time, because the cancer is developing under the basal skin cell layer (Berman, 2011). Because basal cell carcinoma is an anti-metastatic cancer, it will almost never spread to other parts of the body; instead it will stay in one primary location if detected and treated early. Neglecting to take steps in removing the cancerous area opens up the possibility for the cancer to metastasize to local parts of the body, most often the nose, eyes, and ears which will result in the disfiguration of those body parts (Berman, 2011). Basal cell carcinoma of the breast is a cancerous skin growth that is detected on the surface of the breast of an individual. There are numerous methods available today to remove the cancerous growth; these include excision, cryosurgery, electrodessication, and photodynamic therapy. All of these are non-invasive procedures and depending on how quickly the cancer was diagnosed, should permanently remove the cancer. Most basal cell carcinomas do not return once they are removed by surgery or therapy. In the present study performed the basal like cell line MDA-MB-231--- will be used to simulate basal cell carcinoma of the breast. Linking Cataracts to Cancer 3 MDA-MB-231- - - (Figure 2) is a basal-like breast cancer cell line available for purchase at the American Type Culture Collection (ATCC). It is an epithelial based cell line that is found in the mammary gland, or breast of humans. Figure 2. MDA-MB-231 cell line. This is a basal type carcinogenic cell line that will have proteins isolated from it for further analysis. (own photo) MDA-MB-231--- is based on a disease called adenocarcinoma and is a derivative of the pleural effusion of a 51 year old Caucasian female in 1974. This cell line can be applied as a transfection host, and expresses both an epidermal growth factor (EGF) and a transforming growth factor alpha (TGF alpha). These are both very important growth factors for a cell line to have because EGF stimulates cellular growth and proliferation in cells, and TGF alpha is upregulated in cancer and stimulates proliferative cells in the body. MDA-MB-231--- is tumorigenic meaning that the cell line is active and readily forms tumors under any laboratory conditions. The cell line itself is categorized as aneuploid female, with a modal number of 64 and a range from 52-68. The chromosomes N8 and N15 are absent in this cell line, but there are still 11 stable marker chromosomes, some unassignable chromosomes, and a majority of autosomes found in this cell line. MDA-MB-231- - - cells are shown to express the WNT7B oncogene (and also the CRYAB oncogene). The cells are shipped frozen in Leibovitz’s L-15 Medium, and it is advised to produce a new 10% fetal bovine serum so that the cells can thaw properly (“MDA-MB-231”, n.d.). In order to ensure that the cell line is ready for use, the cells need to have the culture medium removed and then need a rinse with a trypsin solution so that latent trypsin inhibitor is removed. New growth media should be pipetted onto the cells and they need to be incubated at 37o centigrade under 5% CO2 conditions until the cells are needed for testing. Not following these instructions to subculture the cells will lead to sources of error in the data because of the trypsin inhibitor. MDA-MB-231--- cells have been used in scientific testing multiple times before, but not specifically for the target protein from this experiment. Triple negative basal like breast cancer lines are normally used so that scientists can acquire a better look at the BRCA1 gene, which is upregulated in the cell line. This means that some of the carcinogenic activity of the cell line comes as a result of BRCA1 gene mutations coupled with other genetic variations. Alpha-basic crystallin is a very recent discovery inside of the cell line; therefore, not much research has been performed on its role in the cell line. MDA-MB-231--- cells are shown to be the second most aggressive basal like breast cancer phenotype when tested in vitro. Other studies performed with the cell line concluded that alpha- basic crystallin expressions in basal like cell lines are associated with poor clinical outcome in breast cancer. This results because the protein induces anchorage independent growth in the cell line, leading to increased cell migration and invasion. Linking Cataracts to Cancer 4 (Moyano, 2006). In order to test that specific qualities occur in only the basal like cell line, another cell line is tested to compare the results of the experimentation. MCF-10A is an immortalized breast cancer cell line acquired from the American Type Culture Collection (ATCC). It is an epithelial based cell line that is found in the mammary gland of the breast of humans. Figure 3. MCF-10A cell line. This immortalized breast cancer cell line is incapable of forming tumors on its own, meaning that scientists use this cell line to look at natural agents that tumorigenic (Debnath, 2003). The cell line derives from a fibrocystic disease isolated in 1984 from a 36 year old Caucasian female. The cell line is also applied as a transfection host and is non-tumorigenic, meaning that the cell line will not readily form tumors unless forced to with the addition of a chemical reagent. This means that the cell line is important when the goal of the testing is researching potential tumorigenic agents. The cell line is produced by long term culture in serum free medium with a low Ca++ concentration. MCF-10A cells are derived from the adherent cells of the culture and the other nonadherent cells are found in different cell lines available from the ATCC (i.e. MCF10F). The cells test positive for epithelial sialomucins, cytokeratins, and milk fat globule antigen. The cell line shows no signs that it is terminally different or that it senesces. The stimuli that the cells respond to are insulin, glucocorticoids, cholera enterotoxin, and epidermal growth factor (EGF). EGF is common to both cell lines used in the testing. Electron microscopy reveals that these cells display characteristics similar to those of ductal luminal cells and not of myoepithelial cells. Reactions tested with monoclonal antibodies of MFA-Breast and MC-5 proteins revealed an expression of breast specific antigens, which is a positive sign for scientists (“MCF-10A”, n.d.). The non-tumorigenic nature of the cell line is important for this testing because the cell line being compared to the basal type breast cancer line needs to be a non-cancerous line. This is because it doesn’t form tumors or add any unnecessary proteins to the testing conditions. Although this cell line is used for a variety of purposes, it is not normally used in comparisons with other cell lines. The primary usage of MCF-10A cell lines, along with similar immortalized cell lines, is to look at different potential tumorigenic agents. These cell lines are considered immortal because they can proliferate indefinitely under proper lab conditions without interruption from other cellular properties. Usually the indefinite proliferation is caused by a mutation or the introduction of some type of oncogene. MCF-10A cell lines have been previously used in laboratory testing to look at such processes as mutagenesis, morphogenesis, and oncogenesis. Linking Cataracts to Cancer 5 Mutagenesis is the origin and development of a mutation inside of a cell line and MCF-10A cells aid in the study of this process by allowing the cell line to be cultured in culture media that contains a frame shift-inducing agent. This biological agent mutates an aspect of the cell line which the researchers can study as the cell line proliferates. Morphogenesis and oncogenesis are commonly tested for together in laboratory testing, where morphological growth agents are cultured into the cell line and then the cell line is exposed to oncogenes in order to disrupt the growth process of these agents (Debnath, 2003). The MCF-10A cell line is extremely versatile and can be used in a variety of ways. Oncoproteins and Oncogenes As discovered through decades of research, oncoproteins play a pivotal role not only in causing a person to develop cancer, but also in helping doctors detect cancer. Oncoproteins are comprised of oncogenes, which are genes that have the potential to cause cancer. Oncogenes are often expressed at high or elevated levels in cancer cells or in tissue cells that have the potential to develop tumors. The term oncogene was coined in 1969 by oncologists Robert Huebner and George Todaro, but the first oncogene itself was not discovered until 1970 when the oncogene src was concluded to be a part of chicken retrovirus. After post-experimentation confirmed the conclusion, the discovery of both new oncogenes and proto-oncogenes (activated oncogenes) lead to current research studies performed all over the world (Mukherjee, 2011). Figure 4. Oncogenetic growth. Oncogenes form from normal cells that contain a proto-oncogene that mutates in some way that causes the cells to undergo neoplastic transformations that change them into cancerous cells (“Oncogene”, 2012). Proto-oncogenes, the basic form of an oncogene, are commonly found in all types of cells in the body and are proven to regulate the promotion and differentiation of the cells. There are multiple proto-oncogenes found in the genomes of cells where they are involved in different steps of the cell’s growth cycle. If there are any alterations in the gene sequence of the cell (caused by the insertion and removal of a retrovirus) or there is an overexpression of a specific protein in that cell, then the proto-oncogenes will cause the host cell to undergo neoplastic transformation (Figure 4) and convert to carcinogenic cells with uncontrolled growth and proliferation. When observing a normal gene found inside the human body, one is also looking at multiple protooncogenes in the DNA of that gene which could mutate at any time. All normal genes have this mutational ability, and more and more retroviruses are causing mutations in the genomes of Linking Cataracts to Cancer 6 genes. This leads to neoplastic transformation and the end result of the development of a cancer cell. There are five other mutations that lead to the transformation of the cells, they are point mutations (hyperactive gene product and transcription proliferator), chromosomal translocation (new location, gene fusion), and gene amplification (Chial, 2008). All of these potential mutations cause the proto-oncogene to transform the host cell. The expression levels of oncogenes and oncoproteins in cancer cells are always found to be much higher than levels found in normal cells. As explained above, when the proto-oncogenes are expressed at elevated levels, they mutate and form tumorigenic and carcinogenic cells. A low expression level of the oncogene means there is no visible damage or harm done to the cells, more so it means that the cell is completely normal and functioning properly (Chial, 2008). Any mutation that leads to the transformation of a cell must first initiate with an abrupt overexpression and activation of a specific oncogene which produces excessive amounts of the coded oncoprotein. The overabundance of the oncoprotein will lead to the stimulation of the cells that signal for the tissue cells to proliferate uncontrollably, this leads to tumor growth and metastasis of the newly formed cancer cells. Without the overexpression of the oncogene there would not be a transformation of the cell, and the proto-oncogenes would stay in a down regulated state, meaning that there is little to no activity shown. There are two states that a gene or a protein can be in at any given time when not at normal levels: up-regulated and down-regulated. When a gene is up-regulated, there are either external or internal stimuli present and they cause the gene to become expressed at a higher level than normal. This occurs in a cell when it is deficient in some receptor, and receptor genes become up-regulated, leading to the formation of more receptor proteins. These proteins then regulate the sensitivity of the cell, and the gene returns to its normal level. The opposite of this process is down-regulation where a gene is decreased in expression by a natural force, and its corresponding protein expression is also decreased. If a cell is overstimulated with a certain neurotransmitter or hormone for an elongated period of time, the receptor genes become downregulated (the corresponding protein expression would also decrease), and the cell stimulation would return to normal levels. When an oncoprotein is down-regulated, there will be a lower expression of that protein. This also means that the gene/protein combination is not as potent, or it expresses the same power in controlling cellular processes as it would if it were at normal levels. The hope for alpha basic crystallin is that the set experimentation parameters will provide the ideal environment for up-regulation of the protein. Alpha- Basic Crystallin Recently, researchers have begun to express great interest in a small heat shock protein called alpha-basic crystallin (αb- crystallin) because of it molecular chaperone like ability when it is exposed to elevated levels of stress. Alpha-Basic crystallin is a member of the crystallin protein family, which are structural proteins that are water soluble and are most commonly found on the surface of the eye lens. These proteins are responsible for maintaining the transparency of the lens and working on nerve regeneration in the eye lens due to injury. Crystallin proteins in the eye lens also work to increase the refractive index of the eye lens while not hindering light progression to the optic nerve. This allows for the individual to perceive objects with a clearer, more defined picture. However, these proteins do not only exist in the eye lens and are referred to as ubiquitous because of their ability to exist in different areas of the body. Areas outside of the eye lens that commonly express crystallin proteins are the heart and the breast, but in the Linking Cataracts to Cancer 7 breast the proteins are often associated with aggressive breast cancer tumors. Under normal physiological conditions, these proteins look and act like normal structural proteins; however, when the protein is exposed to elevated conditions of heat or stress, it begins to resemble molecules called chaperone proteins. Chaperone proteins prevent newly formed polypeptide chains or subunits from aggregating in one specific area of the body. When chaperone proteins are denatured by either elevated heat or cellular stresses, they lose the ability to prevent the aggregation of cellular material. If denaturing occurs, then the chaperone proteins attract other soluble proteins and cause them to aggregate and clump; this aggregation can lead to the development of oxidative nuclear cataracts. Crystallin proteins are split into two primary groups, alpha crystallin and beta/gamma crystallin. While both groups of proteins are ubiquitous in nature and are found in almost all vertebrate organisms, they serve different purposes in the body. Alpha crystallin is further broken up into two subgroups, acidic and basic. Both of the subgroups are found in large aggregates, contribute to cataract formation, and are one of the major chaperone proteins. Beta/Gamma crystallin is usually associated with several metabolic or regulatory factors in the body and does not play a role in cataract formation in the eye. Alpha crystallin, the more molecular chaperone like protein, is also classified as a heat shock proteins, which is transcriptionally controlled and is up-regulated under conditions of heat or stress. When the body receives a heat related stress signal, it undergoes heat shock response which leads to an increased proliferation of those proteins. Each heat shock protein is named for its molecular weight (in kDa or kilo-Daltons), and because the molecular weight of alpha crystallin is approximately 20 kDa, it is referred to as Hsp-20. Heat shock proteins play important roles in cellular maintenance where they can aid in protein folding and intracellular transport (the transportation of other proteins across the cellular membrane). Different heat shock proteins aid the body in different ways; for instance, certain proteins are important phosphoproteins in the heart and aid in smooth muscle relaxation. Other heat shock proteins work in the immune system by binding to specific antigens and help present them to the immune system for removal or acceptance. CRYAB, the oncogene associated with alpha-basic crystallin, is a member of the small heat shock protein (sHsp or Hsp20) family. While alpha basic crystallin (CRYAB) and alpha acidic crystallin (CRYAA) are both chaperone like and heat shock proteins, they are expressed differently and are found in different areas of the body. CRYAB is more ubiquitous then CRYAA, and is found throughout the body of any vertebrate. CRYAA, which is expressed in a 3:1 ratio with CRYAB, is limited to the function of the eye. In basal like breast cancer and metaplastic tumors where there is an elevated level of stress placed on the cells, CRYAB levels are shown to be an average of 83% higher when compared to the normal levels of the gene (Sitterding, 2008). This confirmed the hypothesis that CRYAB is a small heat shock protein, because the elevated cellular stress levels lead to up-regulation in the oncogene and subsequently lead to the up-regulation of the alpha basic crystallin protein. When the gene is exposed to elevated stress or heat levels, whether radiation induced or physiologically changed, it will undergo both up-regulation and denaturation. These changes cause the protein to become expressed at a much higher level than normal. The heightened denatured expression of CRYAB leads to the aggregation of soluble proteins and eventually to the activation of proto-oncogenes, which signals the transformation of the host cell into a cancer cell. Linking Cataracts to Cancer 8 Figure 5. Alpha basic crystallin. This small heat shock protein could be the potential link between cataract formation and breast cancer (“HSPb2”, 2009). Another method for looking at CRYAB and alpha basic crystallin is through phosphorylation, where the addition of aT covalently bonded phosphate group to an amino acid residue location on the protein causes the protein kinase to either become active or inactive, aract amino acid residues that phosphorylation is acts on turning the protein on or off. There are three in proteins: serine, tyrosine, and threonine. Phosphorylation at different amino acid residues leads to different levels of up-regulation or down-regulation in proteins. The most common areas for phosphorylation in CRYAB are on the serine residue points 45 or 59.This phosphorylation is supposed to increase the expression of the alpha basic crystallin in lab tests, although some experts disagree with that theory. Serine-59 phosphorylation of alpha basic crystallin is associated with a down-regulation in the anti-apoptotic effects of the protein, meaning that it would ameliorate the aggressive phenotype associated with the basal-like breast cancer tumors (J. Moyano, personal interview, November 29, 2012). This phosphorylation has no associated effects on the expression levels of the alpha basic crystallin in the breast cancer, those levels remain the same regardless of the phosphorylation location. Western Blot Assay The need to figure out protein concentrations and expressions in cells has been so great in recent years that Western Blot Assays (Westerns) have become a popular method of screening for proteins. Western Blots, otherwise referred to as protein immunoblots, are a method of screening for proteins in a sample of tissue or cells. Westerns use gel electrophoresis, PVDF membranes, and antibodies to separate the proteins based on peptide length and then analyze them using exposures. In order to run a Western, the first step is to lyse the cell lines/tissues with a lysis buffer. The lysed cells are run through SDS-PAGE (sodium doddecylsulfatepolyacrylamide gel electrophoresis) using a polyacrylamide based gel. Then gel electrophoresis is run on the SDS soaked (and negatively charged) cells. After the proteins in the lysed cells have separated based on molecular weight (kDa), the gel membranes are then transferred to PVDF (polyvinylidene difluoride) membranes through a process of either electroblotting or manual transfer. Manual transfer uses filter paper, transfer buffers, and capillary action to transfer the proteins from gel to membrane, but this method is too lengthy (close to two hours) for it to be considered practical in most laboratory cases. Electroblotting is the use of a transfer machine (i.e. iBlot Transfer System) to transfer the proteins from the gel to the PVDF membrane. This method takes approximately seven minutes depending on the machine, and it is more commonly used in laboratories compared to the manual method. Once the transfers are complete the protein blots Linking Cataracts to Cancer 9 are blocked overnight in a solution of TBST (TRIS Buffered Saline + Tween) and diluted antibody. After the overnight blocking is complete, the blots are rinsed with TBST and blocked with a secondary antibody, which is typically conjugated to HRP (horseradish peroxidase). Two more TBST rinses follow that, and then the blots are looked at with an exposure machine to see if the proteins are expressed. The overall Western Assay process can take as few as three days or as much as one week to complete. Antibodies, otherwise known as immunoglobulin, are Y shaped proteins used in the immune system to identify and neutralize any foreign objects that are present. These foreign objects range from harmless bacteria to lethal diseases. The antibody detects the foreign object from its antigen, a unique area that is specialized in every different bacteria or disease, and the paratope of the antibody binds to the epitope of the antigen seamlessly. This causes the antigen to be tagged for destruction by other cells in the immune system. In a Western Assay, the antibody is used to bind to the antigen that it has a similar paratope. An alpha basic crystallin antibody has a paratope that is specifically meant to bind with the epitope of the alpha basic crystallin protein antigen. The binding between the antibody and the antigen means that the protein becomes blocked into the blot while it rotates overnight in a cold room. Once the bound protein is blocked into the blot, subsequent TBST washes can be performed without the worry of the target protein washing off the blot. This explains when the blot is taken to a machine that performs exposures, only the location of that specific protein shows up on the exposure. The antibody is reacting to the chemical reagent looked for during the exposure cycle, and signals that there are bound antigens of proteins at that definite spot on the protein ladder. From the exposed site, scientists can conclude if the protein is actually expressed there. If the protein is upregulated or down-regulated, this means that the protein concentration shown by the antibodyantigen binding is either higher or lower respectively. Gel electrophoresis is a key step in the success of a Western Blot Assay. In gel electrophoresis, cell or tissue samples are lysed for proteins using a lysis buffer and the samples are then loaded into polyacrylamide gel chambers which are submerged in a buffering agent and electricity is used to force the proteins to run down the gel. The proteins, in running down the gel, become separated by their molecular weight (in kilo-Daltons). Smaller proteins (like alpha basic crystallin) will stop running down the gel sooner, while larger proteins will run much further down the gel. The distance that the proteins run down the gel is proportional to the length of time that the electricity is running. The separation of the proteins is critical for a successful Western Blot Assay because if the proteins are not separated by any means, then there would be no easy way to determine which protein is actually being expressed in the assay. Rhodiola Rhodiola is a genus of the family Crassaluceae, which is a family of hardy plants that all store water in succulent leaves. The range for Crassaluceae extends from the northern extremes of the northern hemisphere, to areas in southern Africa where water is scarce. Linking Cataracts to Cancer 10 Figure 6. Rhodiola rosea plant. Rhodiola is only found in high altitudes and in colder regions, and its roots can be used for significant medicinal purposes (“What is Rhodiola?”, 2009). There are 33 genera in the family Crassaluceae, and most of these plants possess no real agricultural significance. The more common uses for these plants are in either herbal medicine or horticulture, where the primary usage for the plants is either as herbal supplements or garden plants. The most common genera of Crassaluceae are Crassula ovata (jade tree), Rhodiola rosea (Rhodiola), and Kalanchoe blossfeldia (Kalanchoe). Rhodiola are often similar in appearance to the related genus Sedum, the stone crops. Often, many authors will merge Rhodiola with Sedum into one genus under the name Sedum. Rhodiola comprises as many as 200 different species, all of which grow in high altitude regions of Asia or Europe. They are often referred to as stonecrops (Sedum) for their ability to grow in even the smallest amount of soil. The most common species of Rhodiola that are used in medicine are R. rosea, R. imbricata, R. crenulata, R. sacra, and R. kirilowii. These plants are the most frequently found and grown species in the wild, and they possess the most desirable sets of natural compounds that can be isolated and used in medicine today. Each species of Rhodiola has its own unique set of phytochemicals that are used in different ways in a laboratory setting. The way to synthesize the nutrients and compounds from the Rhodiola plant is to harvest the root of the plant at the peak growth time (late fall) and then grind it up into a powder. The powder is then suspended in either an aqueous or hydro alcoholic solution (typically of ethyl alcohol) and then stored in a cool place for up to three months or until needed. Another method of working with Rhodiola is to freeze dry the roots of the plant until they are needed to be ground up for use. This allows for the water content of the roots to go down, and therefore they will have a higher concentration of nutrients left over in the root. This method allows for optimal compound synthesis in the lab. Ancient Asian cultures also used Rhodiola for medicinal tea; this method of ingesting Rhodiola is not as nutrient rich, but is still commonly used today in Russia. The other way to ingest Rhodiola is by taking capsules of powdered Rhodiola, which are available commercially. The medicinal qualities of Rhodiola are so numerous and plentiful, that it can be classified as an adaptogen, which is a plant that possesses the innate ability to withstand biological, physical, and physiological stressors. There are currently only seven known Linking Cataracts to Cancer 11 adaptogens in the world which are all found in high mountainous regions in either Tibet or China. Some examples of adaptogens are Panax ginseng (Panax quinquefolius), Siberian ginseng (Eleutherococcus senticosus), and licorice (Glycyrrhiza glabra). Adaptogens are shown to not only help the body fight stress, but also combat other problem areas in the body. Each one of these abilities is controlled by specific compounds found inside of these plants. It is a combination of multiple of these compounds that contribute to the anti-carcinogenic nameplate that they bear. In Rhodiola, the phytochemicals rosin, salidroside, beta-sitorserol, gallic acid, and kaempferol are the main compounds that have anti-carcinogenic implications. These five compounds have also been shown to control the anti-anxiety, anti-viral, anti-fungal, and antistressing abilities of the plant. When a combination of these compounds are exposed to cancer cells, multiple different smaller compounds work at the molecular level to perform tasks such as preventing metastasis, and increasing radiation based death in the cancer cells. The phytochemistry of Rhodiola has revealed that the successfulness of Rhodiola is because of specific sets of compounds. All of the compounds have been grouped and classified under six specific groups: phenylpropanoids, phenylethanol derivatives, monoterpenes, triterpenes, flavanoids, and phenolic acids. Phenylpropanoids are organic compounds that are synthesized from the amino acid phenylalanine. In Rhodiola, an example of a phenylpropanoid is rosavin, which in itself is made up of rosin and rosarin. The presence of rosavin in an extract of Rhodiola has been used to make the determination of whether that extract is genetically pure, with 3% concentrations of rosavin being the standard for a naturally pure extract. Phenylethanol derivatives, like salidroside and tyrosol, are (like the name suggests) derivatives of phenylethanol or phenylethyl alcohol, which is an organic compound that is commonly found in the essential oils of the plant. These derivatives have large antioxidant properties and are the most important compounds for antioxidant therapeutic usage. Monoterpenes and triterpenes are both based on terpenes. The difference between monoterpenes and triterpenes is that the former are made up of two isoprene units in the chemical symbol C10H16, and the latter are made up of six isoprene units in the chemical symbol C30H48. They are both naturally occurring organic extracts that are synthesized from plants. Examples of monoterpenes in Rhodiola are rosiridol and rosaridin, while examples of triterpenes are daucosterol and beta-sitosterol. Flavanoids are ubiquitous in plants and are the most commonly found group of polyphenolic compounds in the human diet. In Rhodiola, flavonoid examples include kaempferol, rodiolin, and tricin. The primary role that flavonoids play in Rhodiola is to act as a response modifier to certain allergens and carcinogens, meaning that they allow for the body to respond in a different, sometimes better, way to a foreign object. Phenolic acids are the sixth group of compounds commonly synthesized from Rhodiola. Gallic acid is the primary phenolic acid synthesized from Rhodiola, and the primary function of it is to serve as an anti-fungal and anti-viral compound. All six of those groups can be synthesized from Rhodiola by a means of fractionation, where scientists take a primary compound, and separates it based on composition of a certain gradient (Khanum, 2006). Research Plan Researchable Question: What is the effect of Rhodiola crenulata root extract on the biosuppression of alpha-basiccrystallin, a novel oncoprotein that serves as a biomarker for predicting premature basal-like breast cancer? Linking Cataracts to Cancer 12 Hypothesis: The protein alpha-basic-crystallin will signal premature basal-like breast cancer, and Rhodiola crenulata root extract will suppress the expression of the protein. Procedure: The cell lines used in the testing were received from the ATCC, cultured into new tissue culture plates, and placed into an incubator. The cell lines were then lysed of their proteins, and a BCA Protein Assay was run in order to determine relative protein concentrations. Gel electrophoresis was then performed to separate the proteins, and the separated proteins were transferred onto a PVDF membrane. The membrane was then blocked in an antibody, and the membranes were coated in a Luminal bath. The membranes were then exposed in a standard laboratory imager scanning for chemoluminescense. The exposures were then analyzed for the protein content using band normalization and pixel density. Methodology: BCA Protein Assay 1.1) 1.2) 1.3) In a laboratory setting, two frozen cell lines were received for testing, MDA-MB-231--(HTB-26, ATCC) and MCF-10A (CRL-10317, ATCC). The cell lines were thawed in a Styrofoam box (12 in x 12in x 12in) filled completely with ice for one hour until the provided culture media defrosted. Inside a standard tissue culture hood (Thermo Scientific) the cell lines were then plated into eight sterile cell culture plates (clear polystyrene, 60mm x 15mm, BD Falcon) for each cell line and were fed with 4mL of primary growth media (Advanced DMEM, Gibco). The plated cell lines were then taken to a cell culture incubator (150L, LEEC GA2000) and were exposed to 37 degrees centigrade temperatures and 5% carbon dioxide levels for 3 days. Every day the cell plates were removed from the incubator, placed into the same tissue culture hood, and fed with 4mL of new growth media (Advanced DMEM, Gibco). Every day prior to the testing of the cells, the cell lines were looked at under a microscope (BA410 model) to ensure that the cell lines were still alive. Using a black indelible marker (Sharpie, fine tip), the plates that contain the MCF10A cells were labeled as follows (there were 2 of each label): 10A-IR-C (Irradiated Control cells), 10ANoIR-C (Non-irradiated Control cells), 10A-IR-Rh (Irradiated Rhodiola cells), and 10ANoIR-Rh (Non-irradiated Rhodiola cells). The process was repeated starting at step 1.2 using the MDA-MB-231--- cell line except on the labels, the 10A was replaced with 231. A 10% ethanol solution was made by diluting ethanol (Fisher, 100%) with 1X Phosphate Buffer Saline, otherwise known as PBS-1X (Cell Signaling Technology) at a dilution rate of 1:10. A 1% solution of Rhodiola crenulata root extract was made by diluting Rhodiola crenulata root extract (1μg/mL, Barrington Nutritionals) in the 10% ethanol solution so that the final concentration of Rhodiola was 100μg/mL. In the same tissue culture hood, plates with the “R” tag in the label received 20μL of the diluted Rhodiola extract, which was pipetted onto each of the plates and then subsequently swirled and mixed by hand. These specific plates were then moved from the tissue culture hood into the same cell culture incubator (at the same temperature and carbon dioxide specifications) and were incubated for fifteen hours. The plates that had the “C” tag in the label received 20μL of Linking Cataracts to Cancer 13 1.4) 1.5) the 10% ethanol solution, and were also swirled by hand. These cell plates were then moved to the same cell culture incubator and were incubated for fifteen hours. After the fifteen hours had elapsed, the plates that had the “IR” tag in the label were removed from the incubator and were taken to a Cesium-137 irradiator (details withheld per FBI measure) and were exposed to 1 Gray of radiation for 20 seconds which served as a simulation of natural stress in the body. After the exposures, the plates were placed back into the same incubator and incubated for an extra three hours. The plates with the “NoIR” tag in the label remained in the incubator for the three hours. After the three hours had elapsed, a clean, used Styrofoam box (12 in x 8 in x 8 in) was filled entirely with shaved ice from a standard ice machine. The cell plates were removed from the incubator and immediately placed on top of the ice (but not into the ice) so that the cells would stay below room temperature throughout the duration of the step. The growth media that remained on the cells was aspirated using a standard laboratory grade vacuum aspirator (AMETEK). PBS-1X was then added in varying amounts (enough to cover the bottom of the cell culture plate) to the plates. The PBS-1X was the swirled using hands, and then subsequently aspirated using the same device as before. This step was repeated twice to ensure that all of the loose cells were properly taken care of. Then 0.5 mL of a RIPA Buffer (cell lysing agent, Cell Signaling Technology, #9806) was added to the cells using a standard micropipettor (200μL) and was left to coat the cells for five minutes. After the five minutes had elapsed, a cell scraper (sterile, BD Falcon 353085) was used to scrape the bottom of the plate so that the cells lysed off of the plate and into the lysing agent. The lysed cells were then transferred into separate sterile microcentrifuge tubes (1.5mL, USA Scientific) kept slightly embedded in the ice using the same micropipettor; the tip of it was changed after every complete transferal. The sixteen microcentrifuge tubes were then brought into a temperature controlled four degree centigrade room, and were placed into a standard microcentrifuge (sixteen tube capacity, Sorvall Micro 17) which was set to 14,000 revolutions per minute for duration ten minutes. After the ten minutes had elapsed, the tubes were placed into a standard centrifuge tube holding rack and were placed into a -80o Celsius freezer (22 cubic feet, U85-22 commercial model) until needed further on in the testing. A BCA Protein Assay Kit (Pierce) was used for this part of the experiment. Solid Bovine Serum Albumin, also known as BSA (sc-2323, Santa Cruz) was diluted into nine specific standards in accordance to the standard test tube protocol instructions included with the kit. In order to figure out the total volume of the working reagent needed for testing, the equation WR= (Standards + Unknowns)*(Replicates)*(Volume of WR per sample) was used. The working reagent was then created by adding 50 parts of Reagent A and one part of Reagent B to a standard 50mL conical tube and then the tube was vortexed using a vortex machine (Corning LSE) for approximately three seconds; the solution turned a green color. Using a 100μL version of the same brand of micropipettor used before, 25μL of all the standards and unknowns were pipetted into three separate wells (none were in the same well) of an eight by twelve sterile well plate (BD Biosciences), the micropipettor tips were changed after every transferal. Then, 200μL of the working reagent was added into each of the wells and the wells were covered and mixed by hand for thirty seconds. Each of the wells that contained either a standard or an unknown mixed with the working reagent was subsequently given 1% TRIS Buffered Saline +tween, referred to as TBST (Cell Signaling Technology, #9997) so that it was a 1:3 Linking Cataracts to Cancer 14 dilution of standard/working reagent to TBST. The well plate was then covered and placed in the same incubator for 30 minutes. After the 30 minutes, the plate was removed from the incubator and allowed to cool to room temperature, at which time the plate was taken to a Microplate Reader (96 well reader, SpectraMax Plus384) and the absorbances of the solutions on the plate were read at 562 nm. The quantitative values that were produced by the machine were copied down onto paper using a standard black pen, and the values were transferred into a Microsoft Excel file. The absorbance values for the standards only were graphed versus the respective concentration of the standard. The values of absorbance were graphed on the x axis, while the concentration (in μg/mL) was graphed on the y axis. A linear trendline (otherwise known as the standard curve) was then calculated using Excel software, and the equation and R2 values were shown on the graph. The equation that was received was then used to determine the protein concentrations of the unknowns. The average absorbance value for the unknowns was given by the Microplate Reader, so for each of the unknowns, the absorbance value was substituted into the equation, and the concentration results were acquired; these results were multiplied by three to take into consideration the fact that the original unknowns were diluted by a factor of three. These values were then converted from μg/mL to μg/μL so that they would compatible with the stacking gel that was made in the next step. In preparation for making the gels, a 10% ammonium Persulfate solution was made using 1mL of PBS-1X mixed with 100μg of solid ammonia (NH3). Western Blot Assay 1.6) 1.7) The stacking gel was made in a different (but same company as before) standard 50mL conical tube using a recipe of 8.2mL of distilled water, 10.0mL of acrylamide (100%, Fisher Scientific), 6.3mL of TRIS (1.0M, 8.8pH, Fisher Scientific), 0.25mL of the 10% ammonium Persulfate solution made earlier, 0.25mL of Sodiumdoddecylsulfate (SDS, 10%, Fisher Scientific), and 0.01mL of N, N, N’, N’- Tetramethylethalinediamine (TEMED, 100%, Thermo Scientific). The same standard 10mL serological pipette as before was used for the liquid volumes above 1mL, except the tip of the pipette was changed after each liquid was transferred. For liquid values less than 1mL, the same micropipettor was used except the tips were exchanged for each liquid. Extreme caution was taken when the acrylamide was used, because it is a neurotoxin. The solution was vortexed for five seconds and then was taken to a gel chamber (Enduro Vertical PAGE System) where the gel was transferred into the chamber using a standard plastic dropper. The gel was transferred into the chamber until it reached a point that was approximately one centimeter below where the teeth of the 1.5 mm gel combs (that went with the gel chamber) would reach to. In order to prevent the stacking gel from drying out, distilled water was carefully layered on top of the gel using a different standard dropper. Care was taken to perform this step slowly so that the water did not combine with the recently poured gel. Standard paper towels were then soaked with faucet water and rung out. These paper towels were then laid over the top of the chamber, and Saran wrap (Stretchtite) was laid over the top of the paper towels. The gel was then left overnight so that it could properly polymerize (this could be done in three hours minimum). The standing gel was made in a similar 15mL conical tube using the recipe 5.5mL distilled water, 1.3mL acrylamide (100%, Fisher Scientific), 1.0mL TRIS (1.0M, 6.8pH, Linking Cataracts to Cancer 15 Fisher Scientific), 0.08mL Sodiumdoddecylsulfate (SDS, 100%, Fisher Scientific), 0.08mL ammonium Persulfate, and 0.008mL of N, N, N’, N’Tetramethylethalinediamine (TEMED, 100%, Thermo Scientific). The same standard 10mL serological pipette as before was used for the transferal of liquid volumes above 1mL, except the tip of the pipette was changed after each liquid was transferred. For liquid values less than 1mL, the same micropipettor was used except the tips were exchanged for each liquid. Extreme caution was taken when the acrylamide was used, because it is a neurotoxin. The saran wrap and paper towels that covered the gel chamber were removed and the gel chamber was rinsed with distilled water from a standard squirt bottle so that any excess acrylamide washed away. Any remaining moisture left in the chamber was then wicked out using the edge of a paper towel. The standing gel was then poured on top of the separating gel until the level of gel reached the top of the chamber. Two 1.5 mm gel combs were then inserted into the standing gel in set spots on the chamber. The gel was left alone to polymerize for two hours, afterwards the gel combs were removed. 1.8) The cells that were lysed at the end of section 1.4 were removed from the -80o centigrade freezer and were immediately placed into a Styrofoam bucket filled with shaved ice. Once the cells had warmed up to just below room temperature, micropipettors were used to transfer ¼ parts of the cells to new microcentrifuge tubes. The amount varied from cell line to cell line because of the calculations that were done based off of the equation results concluded in section 1.5. Once the cells were transferred, the new tubes were labeled appropriately with the same labels as before. 2μL of dye (4X, purple, Thermo Scientific) was transferred into each of the tubes using a micropipettor. Two tubes that would serve as markers for the rest of the tubes were made using 5μL of marking agent (New England Bio Labs) and 2μL of the 4X dye in each. 1.9) The microcentrifuge tubes were brought over to the gel chamber, which had two sub chambers with ten loading chambers in each sub chamber. Each sub chamber was set up in the order of (from left to right): Marker, Blank Chamber, 231CN, 231CI, 231RN, 231RI, 10ACN, 10ACI, 10ARN, and 10ARI. The tag “C” meant it was a control, “R” meant Rhodiola, “I” meant irradiation, “N” meant no irradiation, “231” meant MDAMB-231 cell line, and “10A” meant MCF-10A cell line. The respective proteins were transferred from their tubes to their corresponding chambers using micropipettors, where the tips were exchanged after every protein was transferred. The gel chambers were placed into a larger gel electrophoresis chamber (Enduro Vertical PAGE System), which was then filled completely with an electrophoresis buffering agent (10X, Sigma-Aldrich, B6185) The top was then placed onto the chamber, and the anode/cathode wires were attached from the top of the chamber to an electrical box. The electrical box was then set to 100V and it was left to run for 90 minutes. The chamber worked because of the presence of bubbles in the buffering agent. 1.10) Once the 90 minutes of run time had elapsed, the electricity was turned off and the electrophoresis chamber was opened up. The gel chambers were removed from the electrophoresis chamber and the combination of the two gels were removed from the gel chambers. Using a standard sterilized Exacto knife, the standing gel (the gel found on top of the stacking gel, there was a definitive line between the two gels) was cut away and the stacking gel was slid off of the gel chamber. The electrophoresis worked if the proteins, more specifically the markers, have visibly run down the gel. Two separate standard Linking Cataracts to Cancer 16 containers (the tops to standard micropipettor tip boxes) were filled with enough distilled water to coat the bottom of it, and one gel was submerged into either container. The electrophoresis chamber and all other objects that were associated with the electrophoresis process were rinsed with distilled water and left to dry on a drying rack until they were needed to be used again in the next section (1.11). 1.11) The diluted transfer buffer was made in a 500mL standard graduated cylinder using a recipe of seven parts distilled water, two parts methanol, and one part Transfer Buffer (20X, Life Technologies). Proper safety equipment was an absolute necessity for this step because methanol is a caustic chemical and can be harmful to skin. The diluted transfer buffer was then poured into the rinsed and dried electrophoresis chamber, and the chamber was set aside for later use. On the lab table next to the electrophoresis chamber, eight pieces of standard filter paper (2 in by 4 in) and two PVDF membranes (2 in by 4 in, WestTran) were wet with distilled water, and were set aside until the next step. Then, a gel holder cassette (supplied with the electrophoresis chamber) was opened and was layered with a foam sponge, two pieces of wet filter paper, one PVDF membrane, one of the pieces of gel that has the proteins on it, two more pieces of wet filter paper, another foam sponge, and then the cassette was closed and sealed. The loaded cassette was then placed into the electrophoresis chamber. The second gel holder cassette was then loaded, using the same methodology as the first one, with the other piece of gel. Once this cassette was loaded, it was also placed into the electrophoresis chamber. The electrophoresis chamber was then placed on top of a standard hot plate (Yellowline MAG HS 7, Laboratory Analysis Unlimited) that had a magnet underneath it. A magnetic stirring rod (40mm by 8mm, SPI Supplies, 01405-AF) was then placed into the electrophoresis chamber, and the hot plate was turned on so that the magnetic stirrer spun but the hot plate did not produce any heat. The cover was then placed onto the chamber, and the anode/cathode wires were connected to the electrical box. The box was then set to 80V of electricity for duration of one hour. After the hour had elapsed, the electricity and magnetic stirrer were both turned off, and the chamber was removed from the hot plate. The top of the chamber was removed and both of the gel holder cassettes were removed from it and were opened up. The transferal of the proteins was successful if the proteins have visibly transferred from the gel to the PVDF membrane. The two membranes were then placed into separate containers filled with a blocking buffer (5% BSA TBST) and were allowed to block for 30 minutes at room temperature. 1.12) Two 50mL conical tubes were set up by filling them both with 25mL of a buffering agent (BSA Blocking Buffer, Thermo Scientific). The alpha- basic crystallin antibody (Santa Cruz Biotechnology) was then added to the solution with a micropipettor so that the antibody was at a dilution rating of 1 part antibody to 400 parts buffering agent. Each of the conical tubes were then vortexed for five seconds each, and were placed into a tube holding rack until they were needed later on. The two PVDF membranes that held the transferred proteins were transferred to separate conical tubes (that were prepared in the last step) using standard metal tweezers. The conical tubes were then capped, and then were taken to a temperature controlled 4o centigrade room, where they were placed horizontally onto a rotating machine (360 degree rotation, PTR-35, Grant Industries) that kept them moving constantly. The conical tubes were left overnight to rotate in the room. 1.13) The next morning, the tubes were removed from the rotating machine and removed from the temperature controlled room. * Two containers (same two containers used before in Linking Cataracts to Cancer 17 section 1.10) were filled with lab made 1% TRIS Buffered Saline (TBST), but only enough was used just to coat the bottom of the containers. The membranes with the proteins were removed from the conical tubes using standard metal tweezers and were placed into separate containers of TBST. The containers were then covered with a top, and then were taken to a rocking machine (Labnet ProBlot 25 XL, Spectra Services) where they were placed on the top of the machine, and then rocked at a setting of four for ten minutes. After the ten minutes elapsed, the TBST in the containers was poured out into the sink (the membranes were not removed) and new TBST was poured onto the membranes in the container. The containers were then rocked (at the same setting) for another ten minutes, where the process was repeated once more. This means that the TBST rinse and rock was done a total of three times. After the third TBST wash, the containers were filled with exactly 15mL of TBST, and an anti-rabbit antibody conjugated to HRP (Santa Cruz Biotechnology) was added with a micropipettor so that the dilution rate was 1 part antibody to 3000 parts TBST. Both of the containers were then placed onto the rocking machine for 30 minutes at a setting of 2. This was done to provide gentle motion and to expose the antibody to all parts of the membrane. After the 30 minutes of rocking had finished, the TBST antibody mixture was poured off into the sink, and the TBST rinse process repeated itself for another three times. After the TBST rinses were finished, the protein blots (membranes) were rinsed with 1X PBS in order to remove any remaining bits of extra antibody or residue that might become a source of error. In two separate standard plastic weighing trays, equal parts of Luminal Reagents A and B (Santa Cruz Biotechnology) were transferred onto them using a serological pipette. The amount of the reagent is negligible, just be sure to cover the bottom of the weighing tray. Each of the weighing trays had one of the protein blots placed into them and the trays were placed onto the rocking machine at setting 3, where they were rocked for two minutes. While the proteins were being rocked, a piece of plastic wrap (Saran Wrap) was placed flat on the top of the lab bench. Once the protein blots were done rocking, the blots were moved from the weighing trays to the plastic wrap, where they were stacked from bottom (length wise) to top facing in the same direction as each other. The plastic wrap was then folded over the tops of the protein blots. The blots were then taken to an exposure machine (GBox Scanning Machine), and the machine was set up so that there was a plain white board underneath the blots, and it was scanning for chemoluminescense caused by Luminal. The focus and lighting were corrected so that the exposures would look the best. The exposure times were set for 30 seconds, 1 minute, and 10 minutes. After that, the machine was left to run for the allotted amount of time specified by the exposure times. Once the exposures complete, make sure to save the exposure files to a safe place, and print out copies of each exposure using a printer. These images that were received are where the results were based from, but another test had to be run using an actin antibody to help determine the location of the protein that was just exposed. 1.14) The protein blots were taken out of the plastic wrap and were transferred using tweezers to separate containers that were filled with the same type of buffering agent as used before. The only difference here was that the antibody used was an actin antibody diluted 1:400. The containers were placed onto the rocking machine at a setting of 4 where they rocked for a total of one hour. After 30 minutes, the blots were flipped over. Then the exact same methodology was used starting at the asterisk (*) found in the beginning of section 1.13. After the actin antibody exposures were completed, the exposures were Linking Cataracts to Cancer 18 compared to each other, and a protein ladder was used to determine the exact location (in kilo Daltons) of the protein along with the activity under certain conditions. Results Western assays showed that the concentration levels of the alpha-basic crystallin protein increased in the MDA-MB-231--- cell line when compared to the MCF-10A cell line. The assay also showed that the Rhodiola crenulata root extract had a negative effect on the alpha-basic crystallin concentration levels, which is a positive result. When the Rhodiola crenulata root extract was added to the cells, the concentration level of the protein decreased regardless of the cell line. While the greater portion of the testing produced qualitative results in the form of exposure pictures, quantitative results were extracted from the exposures using a process called band normalization through pixel density. The data was arranged into tables separated by the two different blots, even though later the data would be appended into a final full data table. Each table included the initial pixel density value for the CRYAB target protein, the pixel density value for the Actin protein, the ratio of the proteins, and the normalized CRYAB concentration. The final data table included the addition of standard deviation values along with 95% confidence interval values. Data tables from a preliminary assay performed early on in the experimentation cycle is also included. The BCA Protein Assay was used in the experimental process as a method of determining the amount of gel to make in order to perform successful gel electrophoresis. The results of that assay were completely separate from the actual results of the experiment, but they were pertinent for ensuring successful separation of proteins. Data Tables Table 1. Nomenclature of Tags used to define the different cell culture plates used in the testing. CN231 CI231 RN231 RI231 CN10A CI10A RN10A RI10A Ethanol Yes Yes No No Yes Yes No No Rhodiola crenulata No No Yes Yes No No Yes Yes Irradiation No Yes No Yes No Yes No Yes No Irradiation Yes No Yes No Yes No Yes No Linking Cataracts to Cancer 19 BCA Protein Assay Table 2. BCA Protein Assay Standard Curve Values used in the Figure 7 graph. Concentration 2000 1500 1000 750 500 250 125 25 0 Absorbance 2.038 1.764 1.119 0.930 0.618 0.363 0.245 0.145 0.112 Protein Concentration Concentration (mcg/mL) 2500 y = 987x - 120.96 R² = 0.9926 2000 1500 1000 500 0 0 0.5 1 1.5 2 Absorbance (at 542nm) Figure 7. Protein Concentration as a function of Absorbance in the BCA Protein Assay. This is the graph of the standard curve of the concentration data, the equation shown in the graph is the equation of best fit to determine the assay results. 2.5 Linking Cataracts to Cancer 20 Table 3: BCA Protein Assay Results that were used to determine the proper amount of Standing and Stacking Gel to make for Gel Electrophoresis. CN231 CI231 RN231 RI231 CN10A CI10A RN10A RI10A Concentration 603.00 486.54 465.81 372.05 378.96 508.75 345.40 496.90 3x Concentration 1809.01 1459.62 1397.43 1116.14 1136.87 1526.24 1036.19 1490.71 Average 0.734 0.616 0.595 0.500 0.507 0.638 0.473 0.626 Point 1 0.876 0.683 0.826 0.560 0.464 0.617 0.457 0.488 Point 2 0.591 0.548 0.363 0.439 0.549 0.659 0.488 0.764 Western Blot Assay First Blot Table 4. Blot One results with normalized CRYAB concentration values on the right. Lane 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Average CRYAB 69403 122763 74812 138910 46973 23507 52993 71803 75146 Actin 321169 520209 424323 447844 432316 338424 558339 400279 Ratio 0.216 0.236 0.176 0.310 0.109 0.069 0.095 0.179 Concentration 16239 17733 13249 23308 8165 5220 7132 13480 Ratio 0.017 0.021 0.021 0.027 0.026 0.020 0.029 0.020 30μg 16.584 20.553 21.468 26.878 26.388 19.656 28.952 20.125 Linking Cataracts to Cancer 21 Protein Normalization Ratios using Actin 0.5 0.4 0.3 0.2 0.1 0.0 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I Gel Membrane Lane Label 10A-Rh-N 10A-Rh-I Figure 8. Protein Ratio values for Band Normalization. These are the ratios of the CRYAB concentration to the Actin concentration; these values were used to normalize the CRYAB values. Alpha-Basic Crystallin Protein Concentrations 35000 Protein Concentration (mg/mL) CRYAB/Actin Ratio 0.6 30000 25000 20000 15000 10000 5000 0 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Gel Lane on Membrane Figure 9. Protein Concentrations post normalization for Blot One. Using the CRYAB/Actin ratios, the average Blot One CRYAB concentration was taken and normalized to the values on the graph. Linking Cataracts to Cancer 22 Second Blot Table 4. Blot Two results with normalized CRYAB concentration values on the right. Lane 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Average CRYAB 120044 167168 73301 92564 21659 6465 19270 25588 65757 Actin 353653 344904 326762 363965 372278 274146 463582 376760 Ratio 0.339 0.485 0.224 0.254 0.058 0.024 0.042 0.068 Concentration 22321 31871 14751 16723 3826 1551 2733 4466 Protein Normalization Ratios using Actin 0.600 CRYAB/Actin Ratio 0.500 0.400 0.300 0.200 0.100 0.000 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I Gel Membrane Lane Label Figure 10. Protein Ratios used for Band Normalization in Blot Two. These are the normalized ratios for the CRYAB in the second blot. 10A-Rh-N 10A-Rh-I Linking Cataracts to Cancer 23 Protein Concentration (mg/mL) Alpha-Basic Crystallin Protein Concentrations 35000 30000 25000 20000 15000 10000 5000 0 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Gel Lane on Membrane Figure 11: Protein Concentrations. These are the final CRYAB concentrations post normalization for Blot 2. Combined Blots Table 5: CRYAB concentration results for both blots with standard deviation and 95% confidence values. Lane 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Concentration (Blot 1) 16239 17733 13249 23308 8165 5220 7132 13480 Concentration (Blot 2) 22321 31871 14751 16723 3826 1551 2733 4466 Average Concentration 19280 24802 14000 20016 5995 3385 4933 8973 Standard Deviation 4301 9997 1062 4656 3068 2594 3110 6374 95% Confidence Interval 5960 13855 1472 6453 4252 3595 4311 8833 Linking Cataracts to Cancer 24 Protein Normalization Ratios using Actin 0.6 0.4 0.3 0.2 0.1 0.0 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I Gel Membrane Lane Label 10A-Rh-N 10A-Rh-I Figure 12. Protein Ratios for Normalization between both blots. These are the normalized ratios for the CRYAB/Actin in both blots. Alpha-Basic Crystallin Protein Concentrations 35000 Protein Concentration (mg/mL) CRYAB/Actin Ratio 0.5 30000 25000 20000 15000 10000 5000 0 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Gel Lane on Membrane Figure 13. Protein Concentrations for both of the blots. These are the final CRYAB concentrations post band normalization for both blots are shown on the same graph. Linking Cataracts to Cancer 25 Average CRYAB Concentration 40000 CRYAB Concentration 35000 30000 25000 20000 15000 10000 5000 0 231-C-N 231-C-I 231-Rh-N 231-Rh-I 10A-C-N 10A-C-I 10A-Rh-N 10A-Rh-I Variable Tested Figure 14. Final average protein concentrations for both blots. This graph includes the average CRYAB concentration for both blots with error bars. % Change in Protein Concentration Table 6. Percent Change Tables for the comparison of both cell lines. Average Concentration 19280 24802 14000 20016 5995 3385 4933 8973 Test Variable C-N C-I Rh-N Rh-I Average % Change from 10A to 231 321.58 732.68 283.81 223.07 390.29 Linking Cataracts to Cancer 26 % Change in CRYAB concentration % Change in CRYAB concentration from MCF10A to MDA-MB-231 cells 800 700 600 500 400 300 200 100 0 C-N C-I Rh-N Rh-I Variable Tested Figure 15. Percent Change graph for both blots. This graph shows the difference in the CRYAB protein concentrations between the 10A and 231 cells. Data Analysis and Discussion The overall trend of the data on the protein suggests that alpha-basic crystallin concentration levels are upregulated in the presence of basal-like breast cancer cells. In normal non-cancerous cells, the concentration of the alpha-basic crystallin has a pixel density of approximately 5,800 pixels/mm3. In the presence of breast cancer, more specifically basal-like breast cancer, the pixel density of the concentration of alpha-basic crystallin nearly quadruples to a value of approximately 19,500 pixels/mm3. This suggests that the protein plays a crucial role in the development of the cancer cells through its molecular chaperone like ability under the stressful conditions posed by cancer. When the protein was exposed to elevated conditions of stress through a radiation model, the data showed that the protein became upregulated and therefore more active in the model. This reaffirms the prior knowledge that this protein has behavior similar to that of a small heat shock protein. The addition of Rhodiola crenulata root extract had a small effect on the data, suggesting that it may have minute clinical implications as an anti-cancer agent. The presence of the alpha-basic crystallin protein most definitely has implications as a potential cancer biomarker, especially because of the 390% change in concentration values between the 231 cells and the 10A cells. As shown in Figure 15 below, the exposure of the blots clearly show that the protein appears visibly in just the lanes that contain the MDA-MB-231 cells, and not those that contain the MCF-10A cells. Not only has this protein produced visual results suggesting that it is active in only cancer cells, but also it has quantitatively backed up the visuals with data that shows the protein is much more present in the Linking Cataracts to Cancer 27 breast cancer when compared to normal cells. Statistical analysis revealed that the null hypothesis in two of the three cases could be rejected, which means that the concentration values from the MDA-MB-231 cells are not statistically the same as the values of the MCF-10A cells, meaning that they have an F value that that is greater than the F-critical value provided in the Factorial ANOVA performed on the data. The addition of Rhodiola crenulata showed statistical similarities, and the two blots were not statistically different. Figure 15. Exposure photo looking at the alpha basic crystallin concentrations. This is the final exposure photograph of the proteins. The 4 lanes on the far right are the 231 cells, where the 4 lanes on the left are 10A cells. Conclusions The data that resulted from experimentation supported the hypothesis presented, but it did not fully confirm it. The target oncoprotein, alpha-basic crystallin, was proved to be at elevated concentration levels in MDA-MB-231 breast cancer cells when compared to the immortalized MCF-10A cells. The addition of a stress model also succeeded in increasing the concentration level of the protein. Additionally, the results of the western blot assay also proved that the Rhodiola crenulata root extract had a slight preventative effect on alpha-basic crystallin concentrations. However, unlike the results of the protein levels in different cell lines, the Rhodiola induced cells did not undergo a great enough change in concentration level to be considered significant by any medical means. The significance of the difference in alpha-basic Linking Cataracts to Cancer 28 crystallin concentration levels just between the MCF-10A and MDA-MB-231 cells is something of note to the medical community. Alpha-basic crystallin levels are a viable option to look at in blood testing or urine testing to determine the presence of or stage of breast cancer. Although there were only two data points taken for each testing condition, the trend of the data shows that high levels of alpha-basic crystallin are associated with breast cancer, especially of the basal-like nomenclature. This study is only at the pre-clinical level, but with access to different cell lines and actual clinical patients, this study can be taken in vivo with live testing. Overall, the data concludes that the protein alpha-basic crystallin and its relative oncogene CRYAB possess the high potential for use in the prediction of basal-like breast cancer, a discovery that could potentially save the lives of thousands. Limitations and Assumptions There were some significant limitations that were present in the project including time, location, money, and resources. When it came to working in a lab with cell lines and numerous different chemicals, the experimenter was limited to only the resources that the lab provided for him to use in the testing. The cell lines and antibodies used in the experimentation cost approximately $300-500 each, which meant that the testing was limited to only the materials available without funding. The location of the labs also limited the amount of time spent working in the labs, and the access to that place. The lab used for the testing is located 90 minutes away from home and in order to test on consecutive days, the experimenter had to stay at the home of the Qualified Scientist. This was normal for the experimenter because the QS is his aunt, and he has stayed at her house before. These constraints really limited the scope of the project because without these constraints multiple cell lines and proteins would have been examined instead of just two cell lines and one protein. The controls for testing included ambient air temperature, protein assay kit, incubator temperature, radiation type/duration, freezer temperature, and sterilization technique in both the flow hood and at the lab bench. The ambient air temperature in the lab setting was kept consistent at 22˚ Celsius for the entire duration of the testing, while the incubator temperature was kept at 37˚ Celsius at 5% CO2 concentration when the cells were incubating. In order to keep the proteins from denaturing, it was necessary to use ice to bring down the temperature, and to use a -80˚ Celsius freezer for extended storage. The BCA Protein Assay kit was kept constant during the testing, as well as the radiation treatments for the cell lines that needed to receive radiation. The radiation was 1 Gray of exposure for duration of 19.6 seconds, and a Cesium-137 ionizing irradiator was used for the testing. The control group for the testing was an immortalized breast cell line referred to as MCF-10A. This cell line serves as the closest in vitro simulation for regular breast cells that behave the way that was needed in a laboratory setting. The control group used for the data analysis is the actin protein. This protein was used during the quantification of the data because it is a ubiquitous protein and is accurately used to find ratios in band normalization. The entire project was based on the primary assumption supported by many smaller suppositions. That primary belief was that the alpha-basic crystallin protein existed in the cell lines that were tested. If this were not the case, then the project would have failed and ultimately produced inconclusive results. It was assumed that the laboratory setting where the tests took place was sterile and that proper laboratory sterilization technique was followed to ensure that the data was not corrupted by outside sources. All of the machines that were used in the testing Linking Cataracts to Cancer 29 were assumed to function properly and not produce rogue results. This includes the hemocytometer, gel electrophoresis chamber, iBlot transfer machine, GBox exposure machine, irradiator, freezer, incubator, and centrifuge. It was also assumed that if the machines did not function properly, that a manual option was available to use and that it was just as effective as the electronic method. This assumption was used when the electronic transfer machine broke, and a manual method was used. It was assumed that throughout the course of the testing, that the proteins were kept at ideal conditions and did not denature during testing. Denaturation of proteins would have led to false results for the project. It was assumed that the choice of cell lines and stress model were the most accurate to simulate actual in vivo situations. An immortalized cell line was assumed to provide an accurate lab model of normal breast tissue as compared to any other cell line, and a radiation based stress model was assumed to be more accurate than a heat model. Finally, it was assumed that these results can accurately be extrapolated when similar testing is performed on the same cell lines. Sources of error were small because testing was performed in a laboratory setting under flow hoods and incubators while using sterile technique. However, besides the human error that is unavoidable in testing, other sources of error could include lapses in sterile technique leading to outside contamination, uneven loading of the gel chambers leading to faulty plate readings, and cutting short the amount of time the gel electrophoresis chamber was allowed to run. All of these sources of error lead to minor alterations in the average expressions of the protein’s pixel density, and ultimately to changes in the overall expression of the protein when compared to the control group. Applications and Future Experiments Research is continually performed in the realm of oncology or cancer biology, with the goal of it being to discover methods that could either prevent or treat cancer in people. While the holy grail of all discoveries would be a cure for cancer, focusing in on early detection can be just as effective in preventing fatalities from the disease all together. This is where looking at proteins and protein signaling pathways plays a major role in science. The type of research that is being performed in this project has a high potential for possessing positive implications in cancer early detection and prevention. While being largely attributed to cataract formation in the eye lens, small heat shock proteins such as alpha-basic crystallin, with their ability to aggregate other proteins and then denature them, are a novel target for cancer research. There are currently only a few (less than 10) published papers out about the implications of alpha-basic crystallin as an oncoprotein linked to breast cancer and of the ten papers, only one of those papers pertains to basal like breast cancer. That one paper looked at uses the same control cell line, but the focus of the paper is completely off from the focus of the testing for this project. This means that the conclusions drawn from this project are completely novel and can be used in studies conducted after this testing. Alpha-basic crystallin levels can be found using ELISA Assays, and the specific upregulated level that causes the neoplastic transformation of the cell can be determined. Once this level is determined, it can be used in everyday oncological medicine as a biomarker for premature/developing breast cancer. Future extensions for this project include looking at other cell lines for alpha-basic crystallin concentrations, using different models to simulate stress in vitro, using different testing Linking Cataracts to Cancer 30 methods (for example, immunohistochemistry) to look at different aspects of the protein, and performing in vivo studies in mice. Western Assays are the most basic type of test used to determine the presence of a protein in a cell line, and there are more time consuming and accurate testing methods that could be administered to test for the presence. Also, similar small heat shock proteins can be scanned for to determine if the entire Hsp family has oncological implications. Many other proteins can be discovered by using a genome-wide cDNA microarray, and then those proteins can be looked at further. Anyway it is looked at, oncoproteins are important molecules and should be taken seriously, because one day they could save millions of peoples’ lives. Literature Cited Berman, K. B. (2011). PubMed health. In NCBI. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmedhealth/PMH0001827/ Bueckler, P. (2005). Sarcoma: A diagnosis of patience. Retrieved from http://sarcomahelp.org/articles/patience.html Chial, H. (2008). Proto-oncogenes to Oncogenes to Cancer. Nature Education, 1(1), 1-4. Debnath, J. (2003). Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. In Science Direct. Retrieved from http://www.sciencedirect.com/ HSPb2 (2009). Heat Shock Protein Beta 2 (HSPb2). Retrieved from http://www.uscnk.com Khanum, F. (2006). Rhodiola rosea: A versatile adaptogen. Comprehensive Reviews in Food Science and Safety, 4, 55-63. MCF 10A (n.d.) Retrieved from http://www.atcc.org MDA MB 231 (n.d.) Retrieved from http://www.atcc.org Moyano, JV. (2006, January). AlphaB-crystallin is a novel oncoprotein that predicts poor clinical outcomes in breast cancer. National center for biotechnology information, 116 (1), 261-270. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16395408 Skin Cancer Facts (2010). Retrieved from http://www.skincancer.org/ Mukherjee, S. (2011) The Emperor of all Maladies: A Biography of Cancer. New York, NY: Simon and Schuster Press Oncogene (2012). In Encyclopedia Britannica. Retrieved November 30, 2012 from Encyclopedia Britannica Online: http://www.britannica.com/ Sherr, C. J. (1996). Cancer cell cycles. Retrieved from http://pathology.med.nyu.edu/ Pagano/7.10.07/1672.pdf Linking Cataracts to Cancer 31 Sitterding, SM. (2008, February). AlphaB-crystallin: a novel marker of invasive basal-like and metaplastic breast carcinomas. National center for biotechnology information, 12 (1), 33-40. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18164413 Sweetenham, J. (2009). Treatment of lymphoblastic lymphoma in adults. NCBI, 15(23), 101520. Retrieved on 11/8/12 from http://www.ncbi.nlm.nih.gov/pubmed/20017283 What is Rhodiola? (2009) Retrieved from http://www.perfectacai.com/ReviveResearch.html Acknowledgements The author wishes to thank many mentors for their help during the project. First, he would like to thank all of the people at Pioneer Valley Life Sciences Institute in Springfield Massachusetts for providing moral support and help when it was needed, and for their generous donation of laboratory space and all of the cell lines, reagents, and materials that were used in the project. He would also like to thank Dr. Sallie Smith Schneider for working with the author throughout the course of the project, providing guidance and help when necessary, and for allowing the author to stay at her house during the testing period. He would like to thank his parents for their ongoing support of the topic, and their willingness to provide transportation to and from school and the lab. Finally, he would like to thank Dr. Judith Sumner and Mrs. Shari Weaver for their weekly help with the writing and paperwork for the project.