Breast cancer usually begins in the breast lobules, where milk is produced, or in the breast ducts, which transfer the milk from the lobules. Breast cancer metastasis occurs when a cancer cell leaves its tissue of origin, which in this case is the breast tissue, and migrates to a different tissue type. Metastasis is possible when the integrity of the structures holding the cells in place is compromised.
Figure 1- A) A depiction of the primer placements in the MUPP1 transcript. The arrows represent the direction and approximate location of each primer. B) Gel electrophoresis of the RT-PCR products are visualized on a 1% agarose gel. Each set of bands shows the amplified product from one primer pair in each cell line. The GAPDH gene was used as a positive control.
Figure 2-A) The three different primer pairs and the possible products RT-PCR products they can generate. B) Gel electrophoresis of the RT-PCR products on a 1% agarose gel. Each band corresponds to a possible product depicted in Figure 3A. More preferential splicing of exon 18 can be seen in the MDA-MB-435S, MCF-7, and MDA-MB-468 cell lines than the other cell lines.
Figure 3-A western blot of MUPP1 done by Abcam using .01 ug/ml anti-MUPP1 antibody. There is 50 ug HeLa cell lysate in lane 1, 15 ug in lane 2, and 5 ug in lane 3. The blot shows different sized bands of MUPP1.
Cancer cells become metastatic when tight junction function is compromised. MUPP1 is a protein that is associated with tight junctions and is reported to have decreased expression in metastatic breast cancer (Martin and Wiang 2008). In this project, MUPP1 gene expression levels were examined by RT-PCR in various breast cancer cells. The expression levels were found to be relatively constant between the cell lines. Previously in the literature, alternatively spliced forms of MUPP1 have been detected. The presence of alternatively spliced forms in these cells lines was examined and it was found that they showed varying alternatively spliced patterns. Interesting, none of the splicing events caused a frameshift in the encoded protein. None of the alternatively spliced exons disrupted any of the 13 PDZ domains in MUPP1. An implication of the alternative splicing is that antibodies need to designed so they recognize a conservatively spliced region of the transcript. The role of differentially spliced MUPP1 transcript in the metastatic process needs to be further examined.
I thank Dr. Rubin for helping me with the project, making it possible in the first place, and always steering me in the right direction. I am deeply grateful for all of Catharine Grubaugh’s and Kate Reid's help every step of the way. I thank Dr. Sylvia Anderson for helping me purify RNA. Also, I greatly appreciate Dr. Wei for helping me design the project and for providing the RNA and cells. In addition, I thank Dr. Wu immensely for preparing the cells and helping me with the RNA. Lastly, I thank all of my classmates for their support throughout the project.
|This document was last modified 05/16/2014.|
This site is powered by the versatile Zope platform.
|This is a project of the Biology Department of Fordham University