p27kip1 Protein Levels Reflect a Nexus of Oncogenic Signaling during Cell Transformation.
Cancer can be defined as the disease caused by abnormal cell proliferation in parts of the body or a malignant growth of a tumor resulting from these cells. However, it is not just one disease instead a large group of diseases since the action can happen in any part of the body. There is this ability the cells have to migrate from the site of origin abnormal growth and spread to distant sites. One out of every four deaths in the United States is from this disease which drives scientists to study Cancer Biology and its pathways. In studying the different pathways, there is the hope that we can either make a cure or prevent these pathways from occurring to cause these cells to override the apoptosis. In order to understand the author specific point of research we must first understand SV40-small T antigen, Rb, and p27.
To begin, SV40 is a proto-oncogene which is capable of transforming several of cell types. It seems to disturbed pRb function regulating cell cycle progression, and equivalent to a light switch turning transcription in cells. Compared to pRb functions in cell cycle suppression, it prevents the cell from replicating damaged DNA by preventing its progression of the cell cycle through G1 to S phase. In general, when it binds to E2F it act as a growth suppressor and prevents the cell from going through the cell cycle. Similarly, p27 controls cell proliferation by binding and regulation the activity of Cdks (cyclin dependent kinases, which are responsible for regulation of the cell cycle). p27 is regulated by phosphorylation (much like p53) of tyrosine at the eighty-eighth position in its amino acid sequence by a tyrosine kinase, then triggers CDk2-mediated phosphorylation of T187 and the initiation of the degradation of p27 by the ubiquitination pathway (Wang et al). In summary according to Wang et al, “…cells stimulated to undergo division relieve G1/S arrest through a two-step mechanism of p27 elimination. In the first step, a tyrosine kinase phosphorylates p27 at Y88, restoring partial Cdk2 catalytic activity. With the ATP pocket now accessible for ATP, p27 molecules tethered to Cdk2 are phosphorylated at T187 within their flexible C-termini by Cdk2. T187 phosphorylated p27, within the ternary complex with Cdk2/cyclin A, is now recognized by SCF/Skp2 for ubiquitination followed by p27 degradation by the 26S proteasome. Loss of the p27 protein is a common event in cancer. In addition to playing a role in normal cells, we believe that the stimulation of tyrosine kinases in cancer cells (e.g. chronic myeloid leukemia) provides a mechanism for elimination of the G1/S checkpoint that is mediated by p27.” In short, this protein is a tumor suppressor; its activity stop cell proliferation. Later according to Dr. James Roberts’s studies, the growth phase is halted at G1 preventing cell proliferation, however, if inactivated abnormal cell growth will occur.
The paper seeks to understand what p27 levels reflect in transformed cells and to understand its use to indicate the biological state of the organism. Miller et al, hypothesizes that p27 levels connect with Rb (functions to inhibit cell cycle progression preventing abnormal cellular growth) inactivation coupled to cell proliferation leading to complete cell transformation. In summary, the researcher began the experiment by the following methodology: cell culture and viral infection, immunoblotting, soft agar growth, tumor allografts and analysis of small t antigen and PP2A in p27 phosphorylation. Mouse embryonic fibroblast cells were isolated and cultured then infected with SV40 (contains both the large t antigen and small t antigen) and DL8889 (SV40 mutant containing only the large t antigen) infected human mesothelial cells. Next the cells were grown in media suspended, trypsinized and transferred into dishes of 0.9% agarose. Overall, the cells were infected with SV40 or DL888 for eight passages and the p27 protein had been knocked down; the cells were selected for treatment and tests were performed. In order to determine the amount of SV40, a SDS-PAGE had be done of the extracts from the mouse embryonic fibroblast, then the proteins had been transferred to PVDF membrane and blotted using antibodies. Also in order to see foci formation of the cells, they were plated three times, fed every few days for 14 days and had been stained with crystal violet to see foci that were counted. The researchers had injected nude mice with SV40 and DL888 in order to determine the how tumors formed for 20 weeks or until the animal had to be euthanized according to the size of the tumor (100 cm3). Lastly, phosphorylated p27 had been removed via centrifugation, separated by SDS-PAGE, and the gels were immune-blotted with phosphor-specific antibodies.
From the experiment it seems as though the small t antigen protein of SV40 enhances transcription which inhibits PP2A (protein that all controls cell growth and division) that relates the p27, and its activity depends on p27 and other proteins, may as well. Also it seems that small t antigen was needed to reduce the accumulation of p27 during the human mesothelial cell transformation; in the literature it states that the SV40 is not essential for viral replication. However have these other functions, which include causing cells to go through transcription and grow abnormally. P27 seems to couple with the large antigen portion of the virus to support growth when it is deficient. So in the case of the DL888 , mutant SV40 , the absence of the small t antigen is replace via p27 which leads to cell proliferation because the p27 protein has lost its function. The cell lines did prove to have formation of tumors once they were injected into the immunocompromised nude mice. The SV40 infected and DL888(mutant)-infected by p27 had been able to form tumors while the DL888 wild type had not, showing that the loss of p27 can replace the small t antigen function and the findings also shown that p27 mutation doesn’t affect the expression of the large t antigen. However, the ability of p27 to be phosphorylated at serine 10 and threonine 187 via SV40 is critical for cell transformation.
The findings of the paper suggests that the small t antigen expression can increase the phosphorylation of p27 and the loss of p27 can replace the transformation activity of the small;; t antigen.PP2A contributes to the transformation in the cell signaling pathway and affect p27 which may alter the signaling. It seems as though reduced p27 levels in tumors are good for determining the readout of the cell signaling, and explains its value as a marker for cell proliferation. The data suggests that PP2A activity allows for increase oncogenic signaling through Myc or AKT1 pathway. This activity depends on the environment where the pathways of the two converge with the regulation of p27 which is control by PP2A.This signaling is responsible for cell transformation. Further that when p27 is deficient in the mouse model tumor progress however, when they contain the large t antigen and small t antigen the cancer becomes differentiated and supports p27 and small t antigen as a crucial target for tumor growth as compared to the mice only having the large t antigen, which poorly differentiates into cancer.
Also the study that collected rodent cells are easily transformed compared to a human; and not all cell lines are easily transformed. Due to the complexity of the human genome it takes a lot of genetic events to lead to transformation of human cells. To transform, a human cell requires more than one protein to malfunction. pRb works with p27 for cell cycle inhibition therefore if one is mutated or “out of the question” ,pRb can still function. Nevertheless, p27 plays a role in Myc and ATK1pathways which may contributes to the cell still bypassing the G1 and S phase. The role p27 in the mouse vary when it is compared to a human. Similarly Rb loss is combined, with other genetic events can derive retinoblastoma and sarcoma in human; while in mice it leads to pituitary tumors. This may suggests that specific cell typing for each of the proteins in the mouse model and the differences should be compared to the human to see the difference between the cell’s signaling transduction. The relationship between PP2A and p27 interaction promotes the G1 to S transition. The researcher network suggest, if PP2A is independent of p27 the relationship between the small t antigen and PP2A activity transforming the cell, depends on the strength of the signal. In case for higher levels of the signaling PP2A inhibition would not be essential , because the cells will be able to control the expression of p27 transcription and translation mechanisms. All in all, eliminating ST can replace the role of small t antigen in transforming the mouse embryo fibroblasts and immortalized human mesothelioma cell line and PP2A acts as a tumor suppressor along with pRb, p27 and p53.

Bibliography

Ali SH, DeCaprio JA (2001). "Cellular transformation by SV40 large T antigen: interaction with hostproteins".SeminCancerBiol11 (1):15-23.
< http://www.mcb.uct.ac.za/cann/335/Papovaviruses.html>
"How Does the RB1 Protein Function?”. N.p. Web. 25 Nov 2012. <http://dpuadweb.depauw.edu/cfornari_web/DISGEN/retinoblastoma_website/public_html/protein.htm>.
Roberts, James. "Genes and Development." Genes and Development. N. page. Web. 25 Nov. 2012. <http://genesdev.cshlp.org/site/misc/p27FlipSide.xhtml>
Wang, Yuefeng, Ludger Hengst, Brent Waddell, and Richard Kriwacki. "American Association Cancer Research." American Association Cancer Research. 47.2006 Web.24 Nov. 2012 <http://www.aacrmeetingabstracts.org/cgi/content/abstract/2006/1/1322-a>.