Reseach Proposal

[Miss. Waffle]

I recently wrote my first research proposal and it was approved!＼（＾○＾）人（＾○＾）／ I am just an undergrad at the moment, and I still have a lot to learn. Feel free to peruse my writings over Estrogen-dependent Breast cancer below. I have yet to do any serious lab work since COVID and biology doesn't mix well. Hopefully I can get the ball really rolling in August. Wish me luck, guys.
Undergraduate Research Grant Competition Spring 2020 Localization of G1P3 Protein by Alteration of G1P3 Nucleotide Sequence

ABSTRACT:

Cancer-related deaths are a pertinent area of study in the United States. Statistics show that breast cancer causes the second most cancer-related deaths among American females. Tumor manifestation and propagation is due to unregulated survival proteins. Anticancer drugs are not effective against these unregulated proteins and therefore a poor prognosis is often associated with the disease. The unregulated survival protein to be studied in our laboratory is G1P3. G1P3 is an interferon-induced protein that is present in Estrogen-dependant (ER+) breast cancer tumors and is linked to apoptosis resistance of cancer cells. Studies in our laboratory have denoted G1P3 to be localized inside the mitochondria, and functions to inhibit mitochondria-elicited apoptosis. We intend to validate this localization of G1P3 by altering the nucleotide sequence of G1P3, and then introducing this nucleotide sequence into the mitochondria of an intact living cell. The G1P3 gene will express protein production with cotranscription of an epitope called FLAG. A primary and fluorescently-active secondary set of antibodies will be incubated with the tagged cells, respectfully. Once the tagging process has been completed, a Fluoview 3000 confocal microscope will be used to identify the location of altered G1P3 molecules inside of the mitochondria. This study will develop knowledge of the GIP3 protein location so that future anticancer drugs may be able to accurately target this type of protein.

Background:

Breast cancer claims the lives of ~40,500 women in the United States every year (4) The reach of this disease is said to affect one in eight women annually. A major characteristic of breast cancer is the development of malignant cells in breast tissue. Malignant cells divide uncontrollably as they bypass the normal stages of mitosis. Another observed ability of malignant cells are the release of survival proteins, which attribute to the apoptosis-resistance in cancer cells (l) Known families of unregulated survival proteins Bel- Inhibitor of Apoptosis (IAP) and Heat Shock Protein (HSP) are present in different cancers (6) The unregulated protein family identified in breast cancer is called FAM-14. FAM-14 includes G1P3, a mitochondrial-anti apoptotic protein (2) The G1P3 in turn controls the production of two apoptosis-related proteins, Bel-2 and Bim. Bel-2 decreases apoptosis of the cell. Bim increases apoptosis of the cell. G1P3 operates to inhibit apoptosis by regulating these two proteins to allow rapid cancer cell growth.

G1P3 is exclusively identified as a component of estrogen-dependent (ER+) cancer cells (1) Diseased cells have over-expression of G1P3 via induction of cell interferons (2) A relationship has been studied using meta-analysis comparing the survival rate affect of G1P3 regulation levels in metastasis-free cancer cells and distant metastasis-free survival (3) According to the National Center Institute, metastasis-free survival is a “length of time from the start of treatment for cancer that a patient is still alive and the cancer has not spread to other parts of the body (5)” Cells that lacked normal regulation of G1P3 protein were shown to have a negative relationship to metastasis-free survival. This upregulation of G1P3 is correlated to poor survival rates and an increased probability of cancer cell metastasis. Metastasis is the migration of cancer cells into the bloodstream where tumors can be propagated and spread throughout the body.

Not all functions of G1P3 have been identified, however the amino acid sequence has been documented. Laboratory research conducted in our lab and others has proposed the localization G1P3 in the mitochondria. Through the alteration of the G1P3 nucleotide sequence, which is based on the identified amino acid sequence, a tag epitope may be added to the protein. This will allow specific identification of the tagged G1P3 protein inside of the mitochondria. An epitope is an antigen molecule to which an antibody attaches itself. The completion of this study will reveal stronger evidence supporting the localization of G1P3 in the mitochondria.

HYPOTHESIS:

We hypothesize that the G1P3 protein is localized within the mitochondria, as this is based on previous protein-isolation studies done in our lab. This may be validated by adding a FLAG epitope onto the G1P3 protein via DNA sequence alteration. The cells will have the altered G1P3 nucleotide sequence, now called G1P3-FLAG sequence, introduced into the cell’s mitochondria. These G1P3-FLAG expressing cells will then be subject to a series of incubations with corresponding primary and secondary antibodies. The fluorescent tag is present on the secondary antibody. Therefore, the localization of G1P3 may be proved by the observation of these fluorescent tags during microscopic analysis of the intact cell.

METHODOLOGY:


Synthesis of G1P3-FLAG

Construction of G1P3-FLAG will require designing a new FLAG epitope nucleotide sequence and adding this sequence onto G1P3, creating a G1P3-FLAG nucleotide sequence. This will be accomplished by combining the FLAG epitope and an oligonucleotide, which will become the reverse primer. A PCR reaction will be run using these components. Verification of the G1P3-FLAG amplicon will be operated using an agarose gel (1%) and then extracted. Bam H1 and Hind III restriction enzymes will be used to digest the amplicon and then cloned and transferred into the vector pQCXIP. MCF-7 cells will be transfected with the positive clones. These altered MCF-7 cells will be used in localization studies.

Localization Studies


The MCF-7 cells now containing the G1P3-FLAG sequence will be fixed using paraformaldehyde and subsequently stained with the anti-FLAG specific antibody conjugated with Alexa 488 fluorochrome. DAPI will be used as a costain to allow visualization of the nucleus. MitoTracker Red will also be used as a costain to visualize mitochondria within the intact cell. Microscopic analysis imaging will be achieved using a Fluoview 3000 confocal microscope.


EXPECTED RESULTS AND POSSIBLE PITFALLS:


The expected outcome of this experiment is that the majority of G1P3 protein will be localized inside of the mitochondria. Cloning and immunofluorescence staining are familiar techniques in our laboratory, so no technical issues are expected.

[yoku]

Wow great work thanks for sharing. Really adds a nice dynamic to the dagger discussion.

[nfl69]

an interesting read no doubt, keep us posted on the progress!

other than the potential for improved diagnosis/prognosis/treatment, etc. what are the implications of proving this particular theory?