Breast Cancer

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The aim of the study will be to investigate the functions and roles of "Id-1" and "Id-3" proteins, and to see if their overexpression is responsible for human breast cancer (Jang et al. 2006). Id-1 and Id-3 are part of the Id protein family, which contains all of Id-1, Id-2, I-d 3 and Id-4. Id an inhibitor of DNA binding and differentiation in human cells. (Jang et al. 2006) Id protein also controls a number of cell cycle regulatory DNA. In the experiment, Id-1 and Id-3 will be tested because our hypothesis is that the proteins' overexpression in the body is believed to cause tumor growth and angiogenesis.

Before getting into the details of the experiment, a couple of terms and ideas should be clarified. Angiogenesis is the formation and development of blood vessels. It is normal for this process to occur during fetal development and during the menstrual cycle in females. However if angiogenesis occurs in any other case than the cases mentioned above, this is not normal and could eventually be fatal (Folkman 1985). Cancer cells go through the process of angiogenesis so they can spread and metastasize throughout body and receive the nutrients necessary for their ongoing survival (Margalit et al. 1996). Cancer cells also metastasize by getting into the general blood steam. They are localized in one area and are called "benign" cells. Once they metastasize, they are called malignant which is known as the "human killer" (Dewan et al. 2005).

Breast cancer is the number one killer in female cancer cases, killing over 40,000 women each year (Garger 2006). MCF-7 is the breast cancer cell line most commonly found in patients suffering breast cancer. Since it is the most common cell line, researchers performed an experiment on MCF-7 cells (Jang et al. 2006). At the end of their experiment, they were able to determine that there was overexpression of the proteins Id-1 and Id-3 when MCF-7 cells were present (Jang et al. 2006). These values fluctuate from patient to patient due to the difference in the stage of the cancer and its severity in each patient. The proteins Id-1 and Id-3 were measured using the Immmunohistochemistry technique. The overexpression of these proteins in MCF-7 cells was believed to cause angiogenesis and malignancy of the cells, allowing them to spread throughout the body quickly (Jang et al. 2006). The only way to prove whether proteins Id-1 and Id-3 are causing death in patients with breast cancer is by performing another experiment, which is where my proposal comes about. My proposal is that if we block the proteins Id-1 and Id-3 or prevent them from being expressed, then we will be able to stop the mestastasis of the MCF-7 cells in the body. There are questions that will be raised at the end of the experiment; will angiogenesis stop completely or will it be slower? Will there be any metastasis throughout the body? Can the manipulation of these two proteins lead to a therapy to cure breast cancer? This experiment will be done invivo on nude mice.

The molecular methods that will be used to perform the first experiment are screening expression libraries and producing knockout mice. The methods that will be used in the second experiment will be production of polyclonal antibodies, fluorescent labeling, and autoradiography. For the experiment in general, there will be three groups of mice. In the first group, we will block the genes expressing the proteins Id-1 and Id-3; these mice will be our "knockout" mice (Connor 2006). In the second group of patients, the proteins will be blocked directly by the use of polyclonal antibodies. The third group will be our controlled group to compare with the two experimental groups. All groups of mice will be injected with MCF-7 cells.

For the experiment, we should first obtain a large sample of MCF-7 breast cancer cells because they will be needed to inject in all three groups of mice. We will take a small sample of the MCF-7 cells with a patient affected with breast cancer (Fan and Mendelsohn 2006). These cells will then be cultured in-vitro, hence estrogen-deprived conditions. MCF-7 cells need estrogen in order to proliferate (Fan and Mendelsohn 2006). Instead we will add exogenous TFG-alpha and the cells will proliferate quickly, giving us large amount of MCF-7 cells for the experimental study (Fan and Mendelsohn 2006).

We will begin the first experiment by using "Screening of Expression Libraries". This will first involve the use of the antibodies against the proteins Id-1 and Id-3. In order to obtain the antibodies, the proteins Id-1 and Id-3 are injected into a rabbit. The rabbit's specific immune system will build a response against these foreign proteins or antigens (Benliang et al. 2005). After 2-3 weeks of injection, we remove blood from the rabbit. We will perform centrifugation and remove the plasma from the blood. The antibodies will be found in the plasma. This will be a very timely process taking up to 3 months (Benliang et al. 2005). To ensure that we have enough antibodies for the whole experiment, we will produce as many antibodies possible and will perform antibody cloning if necessary. After getting the specific antibodies, the proteins are synthesized in phage growth and transferred to nitrocellulose membranes by direct contact with plaques. The membranes are incubated with a protein solution to block any non-specific binding sites (Wang and Eden 1996). Further hybridization is done with primary antibodies made against the proteins of interest (Id-1 and Id-3). We will then use a radiolabeled or enzyme linked secondary antibodies that will bind to the primary antibodies. Detection of the protein will be confirmed through Autoradiography (Wang and Eden 1996).

After detection of the proteins, we now know the genes responsible for both proteins of interest by using the DNA library in mice (Baron et al. 1990). After finding the genes responsible, we will try to make knockout mice. We will first gather information the genes we will be working with in terms of size, number of aminoacids, and presence of enhancers in the introns (Connor 2006). Then we will begin to design the construct we want to introduce to the host's genome. From the sequence information and restriction site information, we will be able to determine what plan we will use to make our exogenous gene. The gene will contain a "Neo Selection Cassette", that will knockout the genes of interest in the mouse DNA (Connor 2006).

Cell Migration Gateway February 2006 (Transgenic and Knockout Mouse-Gene Targeting for Beginners)

After building the construct, the DNA should be prepared for electroporation.