Bombas de expulsión y virulencia bacteriana

The colon adenocarcinoma Caco-2 cell-culture model is a well-established model system to investigate cellular differentiation of mature enterocytes in human gut epithelium [175]. In contrast to proliferating Caco-2 cells, the fully differentiated Caco- 2 cells have lost their tumorigenic phenotype during the differentiation process. These phenotypic changes are associated with downregulation of various proteins involved in the cell cycle progression, DNA synthesis/repair and RNA processing as well as a series of distinct biochemical pathways that are involved in protein folding, cytoskeleton formation and maintenance and nucleotide metabolism [190, 191].

Importantly, in the literature, there is a great number of discrepancies in the findings with the use of Caco2 cell model across different laboratories and these inconsistencies might be due to several factors such as the source of cells, the culturing conditions and maintenance and the associated cell passage numbers [192].

Thus, the process of CaCo2 cell differentiation from day 0 to day 21 was investigated via cell staining for a brush border enzyme alkaline phosphatase (AlkP). In addition, it has been shown that ALkP acts as an important indicator during the different phases of enterocyte differentiation as it is highly expressed in fully differentiated Caco2 cells [177]. In agreement with the literature, confocal results (figure 2.7 and 2.8) demonstrated that the expression levels of AlkP is increased in the CaCo2 cells when they were in culture for 14 to 21 days, in relation to the first and third day of culturing, verifying that the CaCo2 cells portray differentiated phenotype from day 14 and onwards.

Figure 2.7: CaCo2 cell differentiation from day 0 to day 21 was investigated via cell

staining for alkaline phosphatase (AlkP). Top panel shows the red staining fluorescence of

Caco2 cells indicating the distribution of AlkP levels whereas the bottom panel is an overlay of the DAPI (blue) nuclear staining and AlkP staining. Images were captured with a Zeiss

confocal microscope by using mCherry and DAPI filter sets and analysed with Zen (blue edition) Image software. Scale bar at 20 µm, n=2 per time point. Images taken at 40x

magnification.

Figure 2.8: An approximate quantification of the red fluorescence intensity during

CaCo2 cell differentiation from day 0 to day 21. Region of Interest (ROI) were drawn to all

were not stained. Images were analysed with Zen (blue edition) Image software. Means of n=2 with standard error (SE) are shown in the graph.

Additionally, an approximate measure of the mean red fluorescence signal was obtained with the use of Zen (blue edition) software and displayed a steady increase of red fluorescence from day 0 to day 21 (figure 2.8). Moreover, from confocal imaging analysis (figure 2.7), it became apparent that the differentiated Caco2 monolayer started to lose the ability to retain the tight junction formed between the cells after the 15th _day of cell culture. This was evident by observing empty spaces formed at the bottom of the well where a uniform cell monolayer existed previously. Therefore, it was decided that caco2 cells were allowed to differentiate for a period of 14 days prior to start the serum starvation and subsequent serum stimulation time series experiments.

Differentiated Caco-2 cells have been widely used in several studies to investigate important cellular processes that have altered in human colorectal cancer [65, 193, 194]. However, there is no published research article in the literature demonstrating the use of differentiated Caco-2 cells to investigate the protein oscillatory capacity in human intestinal epithelium systems.

Figure 2.9: SOCS3 protein profiles in differentiated Caco-2 cells from overnight serum

free medium. (A) Immunoblots of β-actin and SOCS3 kinetics in differentiated CaCo2 cells

expressed as a fold change in relation to t=0. Means with standard error (SE) are displayed for each time point. Blots and graphs are representative of 3 independent experimental replicates

(n=3).

For the first set of time course experiments, fully differentiated Caco-2 cell cultures were incubated with serum free medium overnight to allow cell synchronization prior to serum stimulation. The control time point 0 was not subjected to serum stimulation during the time course. The results from the time course experiment in fully differentiated Caco-2 cells are presented in figure 2.9.

The immunoblot images from the western blotting experiment (figure 2.9A) did not display any visible clear oscillatory pattern in the abundance of SOCS3 protein during the 8-hour time course in differentiated Caco2 cells. Even though, the bands of β-actin protein show an equal protein loading across the experimental replicates, the above results fail to establish the presence of any type of dynamic behaviour of SOCS3 after serum stimulation.Furthermore, the western blot analysis displaying the fold change of SOCS3 levels in relation to t=0 (figure 2.9 C) demonstrated that the abundance of SOCS3 protein showed a minor fluctuation in the time period of 8 hours. However, the data failed to generate peaks with values 1-1.5x fold higher than the lowest signal values during the time course, consequently the results can’t be considered as a statistically significant.

The absence of the oscillatory capacity in the differentiated Caco-2 cell model system demonstrates that the above cell culture conditions prior to serum stimulation was inadequate to display any meaningful dynamic behaviour of SOCS3 protein. Therefore, to further attempt to establish an oscillatory pattern of SOCS3 protein, the serum starvation of the differentiated colorectal Caco-2 cell line was further optimized.

Considering carcinogenesis as a robust system, which retains its robustness capacity by redundancy through the genetic heterogeneity of cancer cells and through feedback loops that include intracellular and host-tumour interactions [195], it is important to maintain cellular stress at a minimal level in order to avoid false findings resulting from the starvation process and achieve the maximum cell synchronization in culture. Thus, the differentiated Caco-2 cell line was serum starved for 36 hours prior to serum stimulation. The data are presented in figure 2.10.

Figure 2.10:SOCS3 expression profiles in differentiated Caco2 cells from 36 hours

serum free medium. (A) Western blot analysis of SOCS3 protein levels (n=3). (B) and (C)

Quantification of SOCS3 levels in fully differentiated Caco-2 cells was quantitated, normalized and graphed against a linear time scale (n=3). Means with standard error (SE) are

The immunoblot figures from the western blotting experiment (figure 2.10A) demonstrate clearly that there is no oscillatory pattern in the abundance of SOCS3 protein during the 8-hour time course in differentiated Caco2 cells. It is clear from both the immunoblots and graphs that quantified SOCS3 levels against β-actin levels and normalized to the first-time point (t=0), the period of serum starvation and subsequently serum stimulation does not play an important factor in elucidating an oscillatory behaviour of SOCS3. Additionally, this concluding observation is strengthened by the low values of errors bars across the time points in the time course.

However, this report failed to detect any consistent dynamic behaviour of SOCS3 with serum stimulation. This behaviour could be explained by a weak auto-inhibition in this specific cell line, or a quick desynchronization of cell population after the addition of complete medium.

The initial objective was to establish a synchronous cell population. Despite cell cycle synchrony, molecular processes of several proteins are not dependent on cell cycle status. Peripheral circadian rhythms are thought to impact on cellular ultradian cycles and these can be entrained by serum shock [181].

Therefore, to further characterize this system, this report attempts to find other methods to synchronize the cell population and elicit the rhythmicity of SOCS3 protein abundance and study its functionality.