P53 is a tumour suppressor, it controls tumorigenesis via cell arrest and apoptosis. In the early 1990s Murine Double Minute 2 (mdm2) was associated with p53 interaction. Oliner et al. 1993 reported the interaction of the p53 with the N-terminal on the mdm2, caused the inhibition of p53 transcriptional activity. Manfredi 2010 showed that in the C-terminal mdm2 consists of a ring finger, giving mdm2 ubiquitin protein lingase (E3) properties, causing degradation of p53 through proteasomes. Taken together, these observations qualify mdm2 as negative regulator of p53. Donner and Mayo 2001 demonstrated that phosphorylation of mdm2 at serine166 and serine186, encourages mdm2 relocation from cytoplasm into nucleus. In nucleus, mdm2 forms a complex with p53, subsequently causing its degradation.
What are the major findings of the study?
Hung et al 2010, reported activation of mdm2 by Akt phosphorylation, and the adverse effect on p53 transactivity. In my opinion this study demonstrates and provides further evidence in vivo, that activated form of mdm2, causes inhibition of apoptosis via p53 degradation in mammary epithelium cell (MEC) during involution. Over expression of mdm2 localised in the nucleus diminishes p53 levels by mdm2-dependent p53 degradation, thus resulting in accelerated tumorigenesis. Considering all of the evidence, it is clear that Akt-mdm2 interaction encourages inhibition of p53 mediated apoptosis, thus promoting malignant pathophisiology in vivo.
Summarise the experimental approach?
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Transgenic mice were generated, using sub-cloning to introduce cDNA encoding mdm2 and mdm2dd into the mouse mammary tumour virus (MMTV) promoter. This allows the authors to examine the effect of mdm2dd on p53 mediated apoptosis, in comparison to mdm2 and control mice. This transgenic method is considered "gold standard". In contrast there is no control over gene integration and gene expression. Gene expression was measured, at RNA level using transcription PCR, and at protein level using immunohistochemical staining and immunoblotting. In measuring transgene expression, it allows the authors to determine the affect which mdm2dd- p53 relationship has on apoptosis and tumourigenesis. Increased mdm2dd expression leads to delayed apoptosis and an enhanced rate of tumourigenesis due to p53 degradation by mdm2dd. Whole mount and staining techniques were used to observe morphological variances in MECs of control and mdm2dd mice. Apoptosis was measured quantitatively by TUNEL assay, at RNA level by measuring expression of Tweak, LIF and TGFb3 genes associated with inducing apoptosis. These allow the author to compare apoptosis in control mice and mdm2dd mice, and conclude how mdm2 affects apoptosis and its rate by inhibiting p53 activity. A t-test, allows the authors to statistically prove that phosphorylated form of mdm2 slowed down apoptosis and speeded up of tumorigenesis.
How have the authors substantiated the findings?
There is no evidence in suggesting that the authors have repeated the experiment to verify the results. However their findings are consistent with previous investigations Hung et al. 2001. In my opinion the experiment provides in vivo evidence to the current data, and continues to support previous investigations carried in vitro. Application of the T-test is statistically viable when comparing the levels of expression for mdm2dd and p53, in control and transgenic mice, however it relies on assumptions. In answering their hypothesis, the study uses several methodical approaches. Gene expression was measured at RNA level utilising transcription PCR, and also at protein level using immunohistochemical staining. Apoptosis was measured using TUNEL assay, and also by measuring Tweak, LIF and TGFb3 genes expression associated with apoptosis in mammary involution. Bitransgenic mice were generated to explore the possibility of mdm2 association with other oncogenes in tumorigenesis. The results are significant in proving the objective of the study, but there is no ground breaking evidence in better understanding Akt-mdm2-p53 pathway.
How could the study be improved?
The transgenic method applied is generally accepted amongst scientists, one way to improve the method would be to have a control over gene integration and expression. The immunohistochemical staining used can cause background staining which can interfere with result interpretation. Vosse et al 2007 found that by using HRP labelled polymer no background staining was observed. TUNEL assay can give false positive or false negative results. In order to achieve a high degree of accuracy, stains should be carefully prepared, and more assays performed. The experiment can be improved by repetition to verify the findings, assessing the effect of mdm2dd in different tissue type.
How has the study changed the understanding?
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In my opinion this study doesn't take existing knowledge any further. On the other hand it builds upon current knowledge by providing in vivo evidence. It seems that this study has taken relevant conclusions from various literature associated with phosphorylation of mdm2 by Akt and its effect on p53 activity, and tested them in one experiment. These findings were already established prior to this investigation. Moreover it doesn't look in detail the molecular mechanisms involved, it only provides an overview. Authors do not test a new concept, to take the research to a new level.
Have the authors achieved their goals?
The authors have addressed the hypothesis appropriately. This is reflected by the methodical approach taken. By measuring the apoptosis in MECs, the authors prove that mdm2dd leads to delayed apoptosis. Overexpression of mdm2dd in the nucleus caused depletion of p53, resulting in increased rate of tumour development. Conclusively these two measurements reiterate abnormal pathophysiology in vivo. On the other hand, why address a theory when it has already been proven. In my opinion there is no evidence that this study exceeds it objective, it only provides in vivo conformation supporting the current literature.
What are the next questions?
The first question would be how to prevent mdm2-p53 complex, so that p53 can express its transcriptional activity? How to inhibit ubiquitin lingase activity, and phosphorylation of mdm2? Perhaps modify mdm2 or p53, so that p53 degradation doesn't occur. Consider other pathways, binding of p14ARF with mdm2, limits the negative regulation of p53. Ribosomal protein RPL26 enhances p53 expression (Manfredi 2010). Exploration of these molecular pathways may lead to an outcome which is clinically relevant in the treatment and prognosis of breast cancer.