By compilation of available evidence, Yook et al state that through hyperactive Wnt signalling, β-catenin-T-Cell factor [TCF] triggers epithelial mesenchymal transition [EMT] in human breast cancer cells. They then hypothesise that canonical Wnt signalling can result in tumour cell dedifferentiation and tissue-invasive activity – through an Axin2-dependent pathway. They aim to demonstrate this can occur by the stabilisation of the zinc transcription factor, Snail1 that they state plays an imperative regulation role of EMT programmes.
Comment: The context of the experiment including present-day scientific evidence and present-day understanding was well established on the onset. However, the opening paragraph is a somewhat convoluted blend of the author’s experimental aims supported by an array of scientific facts. It’s hard to discern which of these were deduced prior-to and which post- their experimentation. A description of the experiments and hypotheses being tested: including the controls used, number of experimental replicates and the statistical analysis used. A critique of how effectively the data is presented. Finally, does the experimental data really show what the authors say it does?
Figure 1 is subdivided into four panels: 1a-1d – each visually recording results of experiments investigating particular questions that collectively challenged the hypothesis to test its validity.
The Term Paper on The Microscope Experiment 1 Cells
THE MICROSCOPE EXPERIMENT 1 BACKGROUND: The use of a microscope is to provide a magnified view of objects (that are being analysed) that are otherwise to small to be seen by the naked eye. They can be described according to their illumination and lens arrangement. (i) Microscopes are able to use either light or electrons as their illumination source, which are respectively known as light powered ...
Figure 1a shows the confocal microscopy results of mock-transfected fluorescently-labelled MCF-7 cells and Wnt expressing tumour MCF-7 cells cultured on the vascularised epithelial-stromal tissue of the chorioallantoic membrane [CAM] of 11-day-old chick embryos. The authors hypothesised the mock-transferred MCF-7 cells would remain confined to the upper CAM surface as they were unable to express a tissue-invasive phenotype as expected with the Wnt expressing tumour cells [able to invade the stromal matrix]. These two variants acted as the control and tested cell lines respectively. Both the invasion and depth of invasive front were recorded using a mean of three repetitions taking into account the standard error of the mean [representing the spread of the mean of the sample i.e. an indication of how accurate the mean is]. To test the true statistical significance however, the P value was also shown – providing clear quantitative data. The advantages of confocal microscopy were used maximally – it was easy to distinguish between cells, distinguish the CAM surface and follow the points of invasion through. Even more so, the proposed down regulation of E-cadherin by the cancerous cells can be seen extremely well by the loss of the red-occupied area. One may argue, however, that the images showing β-catenin shifting into the nuclear zone aren’t very clear as the nuclear border hasn’t been defined in these single-colour images – somewhat questioning their conclusion drawn from this.
Next the authors aim to show that Wnt-1-transduced MCF-7 cell activity is linked to increased Snail1 protein levels as well as the repression of E-cadherin transcription. With actin included as a loading control, a section of a western blot is presented clearly showing the induction of nuclear Snail1 proteins in MCF7-Wnt cells [compared to the mock]. Only the best results have been presented and no repetitions are shown. Representing the down-regulation of E-cadherin, a bicolour vertical bar chart with confidence intervals is plotted – again recorded using a mean of three repetitions taking into account the standard error of the mean. This shows that the authors recognised the underlying uncertainty – failure to do this would likely lead to misunderstanding and bias, thus reducing data reliability. However, it is unclear why E-box mutants were included in this graph as there was no subsequent discussion about these results.
The Term Paper on Effects of Tonicity on Cell Membrane
Abstract The purpose of this experiment was to determine the effects of tonicity on a cell membrane using red blood cells, potato strips and three unknown solutions (A, B, C). First three slides were prepared containing RBC’s and unknown solutions A, B and C. A control slide was prepared only using RBC’s. After observing each slide under the microscope it was determined that unknown solution A was ...
To determine whether the Wnt-initiated EMT programme can be mediated by the canonical pathway, MCF-7 cells were transfected with a dominant-negative TCF-4 construct. This was an extremely effective method as the gene product would adversely affect the wild-type gene product. As expected E-cadherin repression was relieved and invasion completely suppressed. Diagrammatically this is clear using another confocal-microscopy image that allows direct comparison to the MCF-7-mock result to deduce that in fact Wnt-TCFDN conditions mimicked the MCF-7-mock.
Figure 1c boasts all the same qualities of image 1a – but contains nice use of asterisks to differentiate between the P-values versus mock/siRNA experiments. The diagram effectively supports the hypothesis that β-cateninS33Y-induced CAM invasion is blocked by Snail1 siRNA or shRNA. Although the confocal-microscopy images support this case for siRNA, there is no data provided to support the shRNA claim made. The controls contained no RNA and again the mean of three repetitions was taken.
Figure 1d shows similar analytical techniques to those discussed in Figure 1a and b and supports the hypothesis that S33Y-transfected cells reduce E-cadherin promoter activity, and, that silencing of Snail1 by si/shRNA inhibits the invasive activity of these cells thus restoring E-cadherin promoter activity. Together with the mock, we see a lane for the β-cateninS33Y control siRNA with clear immunoblots showing the up and down regulation as hypothesised. Unlike diagram 1a we are provided with uncropped blot images in the supplementary sections. What are the author’s conclusions at the end of the paper. In your opinion, does the data presented justify this conclusion – why? Suggest two experiments that could be done to support the conclusion.
Describe a question that this paper raises that you find interesting.
Initially the paper mentions that the molecular mechanisms, by which the β-catenin-T-cell factor complex induces EMT-like programmes, remain undefined. The pathway is of fundamental importance and can be used to gain a much deeper understanding into the effects and actions of hyperactive Wnt signalling – particularly in cancer therapeutics [such as in breast cancer] where EMT programmes characterise a tissue-invasive phenotype encouraging metastasis – thus is extremely interesting!
The Term Paper on Cell Cycle
?The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to its division and duplication that produces two daughter cells. In cells without a nucleus, the cell cycle occurs via a process termed binary fission. In cells with a nucleus, the cell cycle can be divided in three periods: interphase—during which the cell grows, accumulating nutrients needed for ...
The authors concluded that the canonical Wnt-dependent regulation of Axin2 does in fact function as an important axis for regulating EMT programmes in cancerous states and thus proved that their hypothesis was correct. For several reasons I believe the data presented does justify this conclusion. All experimentation was conducted very thoroughly using a huge array of different techniques and outlined in the ‘methods’ section and recorded data, generally, was presented very clearly with supplementary information showing a lack of bias.
The invasion activity of non-functional mutated Wnt can be observed on CAM cultured MCF-7 cells simply to confirm the fundamental basis that EMT/invasion will only occur if Wnt is present. A control with actin can be used instead of Wnt. If the mutated, non-functional Wnt experiment induces invasion then the linearity of this experiment will be proven flawed as other signalling pathways are inducing EMT – thereby invalidating all the data collected.
To confirm that the presence of Axin2 is vital in EMT generation [as it controls GSK3β levels in the nucleus thus regulating Snail1 ubiquitination and degradation, i.e. stability] we can transfect a cell line with Axin2, si/shRNA and Snail1 using a control containing non-silencing siRNA. We will expect that EMT will not be initiated when Axin2 is silenced – as GSK3β-dependent phosphorylation of Snail1 will occur in the nuclear compartment. Non-silencing siRNA [control] will allow Axin2 interaction with GSK3β, thus confirming our hypothesis.
