02 Apr 2018
One of the essential function of an organism is to maintain its genome integrity, in order to survive and pass on its genetic materials to the next generation. However, as the carrier of genomic information, DNA is constantly being damaged by exogenous and endogenous factors. Therefore, through evolution, a variety of mechanisms that counter the effect of DNA damage emerged. In this study, we applied different dosages of UV light and Cisplatin, as DNA-damaging agents to SV40 T antigen immortalized MRC 5 Cells, to examine the various cell responses by assaying the relative concentration of key cell cycle regulatory proteins.
In human body, DNA cells carried are constantly under assaults by exogenous and endogenous agents, causing a significant amount of lesions per day (Lindahl et al., 2000). If those lesions are not treated efficiently and the accumulation of lesions continues, mutations and aberrations of genome may lead to the emergence of many forms of physiological disorders, such as tumorigenesis, Parkinson's disease and Friedrich's ataxia (Stratton et al.,2009; Mirkin et al., 2007; Yang et al., 2008).
One of the universal exogenous DNA damaging agent is ultraviolet(UV) light. UV-A and UV-B are its residues that are not absorbed by the ozone layer, and these residues may induce approximately 104 lesions to an exposed cell per hour (Doll et al., 1981). UV light is both a mutagen and potent cytotoxic agent ,which can trigger cell apoptosis by either accumulating DNA lesions or trigger CD95/Fas receptor and induce apoptosis directly (Rehemtulla et al., 1997).
Another well-characterized DNA damaging agent is cisplatin. Unlike UV light, It is usually introduced to the human body during chemotherapy. (Prestayko et al., 1979) Researchers had already identified its DNA damage mediated apoptotic signaling pathway, which involves the TAB1 regulation of proteins' circuitry (Yan et al., 2013).
While DNA is damaged within a cell, there are many possible cellular responses, such as RNA processing, cell arrest and repair. DNA damage repair mechanisms varies according to the type of DNA damage. Apoptosis can be induced by accumulative DNA lesions to ensure the integrity of genome (Stephen et al., 2009) .
Materials and Methods (Instructed By Practical Handout)
SV40 T antigen immortalised MRC5 cell cultures
SV40 T antigen immortalised MRC5 cells were split into eight experimental dishes with the appropriate medium. Those dishes were then incubated (at 5% CO2, 37oC) for two days and ready for cell damage and protein assay.
UV light and Cisplatin were employed as DNA damaging agents. For UV light, we chose 4 levels of dosages:64 J/m2 , 125 J/m2 , 250 J/m2 and 500J/m2; as for cisplatin, we choose to pipette 6.25uM, 12.5uM and 25uM to cells. All cells are damaged for 22 hours.
Cell harvesting and protein assay
After 22h, cells were harvested and prepared for protein assay. Immunohistochemistry and fluorescence microscopy were employed to indicate the level of proliferating cell nuclear antigen (PCNA), and western blotting technique were used to indicate the level of cyclin D1, geminin, tubulin and γH2AX for eight cell cultures separately.
Hypothesis, Mechanisms, and Expected Results
The hypothesis being tested were: For a cell culture, the overall type and magnitude of DNA damage response depends solely on the level of DNA damage they receive. According to our theory, while cells receive endurable damage, DNA repair mechanism will be favored statistically, which results in cellular arrest and DNA repair. Whereas, when the DNA damage level exceed a threshold value, cells will tend to proceed to apoptotic pathway.
Since we assume there can only be two types of cell fates: cells suffered DNA lesions will either activate the arrest/repair pathway or they will activate the apoptosis pathway. Therefore, by protein analysis on certain key cell cycle regulatory proteins that are involved in those two pathways, we can see the relative level of protein expression and hence deduce the cell responses at different levels of cell damage.
Here we choose to radiate UV light and pipette cisplatin at various dosages to different cell cultures. For UV light we choose 64 J/m2, 125 J/m2 as low dosages and 250 J/m2 and 500J/m2 as high dosages. As for cisplatin, we choose 6.25uM as low dosage, and 12.5uM and 25uM as high dosages.
During this experiment, we examined the level of four proteins via immunoblotting: the first protein indicator we examined was cyclin D1. It is a protein that can be activated in the arrest and repair pathway. Previous investigations also suggest it will prevent low dosage UV-induced apoptosis and promotes adaptive resistance (Ahmed et al., 2008).
The second protein we assayed was geminin. It was initially identified as an inhibitor of DNA replication (McGarry and Kirschner, 1998). Later investigations identified an increase in geminin level are correlated with increased proliferation of tumor cells, suggested that geminin may play a role in tumorigenesis (Wohlschlegel et al., 2002, Montannari et al., 2005). It seems contradictory though, overexpression of geminin in the Drosophila embryos leads to ectopic neural differentiation and cell apoptosis (Quinn et al., 2001). We believed, the functional role of geminin varies according to their presence in the different phases of the cell cycle. Under normal conditions, the majority of geminin will concentrate in G1 phase to regulate cell proliferation efficiently. Whereas, under pathological conditions, geminin will tend to present in S or G2 phase, where it has little influence on the regulation of cell proliferation.(Shreeram et al., 2002, Yoshida et al., 2004).
The third protein we examined was tubulin. It plays a vital role during mitosis, and it is responsible for the production of spindle fibers in the metaphase (Wang et al., 2014). In this case, tubulin is an indicator of cell proliferation and apoptosis levels.
The last protein we examined via western blotting was γH2AX, which is a sensitive molecular indicator for DNA damage repair. Because, when a double strand break is detected, one of the early cellular response will be the phosphorylation of H2AX, leads to the formation of γH2AX (Mah et al., 2010) .
We also employed immunofluorescence technique to examine the level of PCNA in cells suffered DNA damages. Previous studies indicated that while UV light is inducing DNA lesions, p21 will be degraded, induce PCNA activation which then leads to DNA repair. (Soria et al., 2006)
According to the functional mechanisms of different proteins, we would expect to see a relatively higher expression level of geminin,γH2AX, cyclin D1 and PCNA while a cell culture is damaged with low dosages of genotoxic agent, and vice versa. Because theoretically, these proteins are involved in cellular arrest and DNA damage repair responses. We would also expect the level of tubulin to decrease gradually while cell damaging level is increasing. Because DNA damaging agents can induce apoptosis and decrease the tendency of cell proliferation.
The actual outcome of this experiment was astonishing. On the one hand, we acquired a relatively consistent green immunofluorescence assay result (Fig 1). The control dish shows a constant level of PCNA expression. When a low dosage of cisplatin was applied to the cell culture, the expression of PCNA tends to increase (Fig 1b), which suggested more cells were undergoing DNA repair. So far our experimental results are consistent with previous researches (Stelter and Ulrich, 2003; Soria et al., 2006). But, when 12.5uM of cisplatin were applied to the cell culture, the level of immunofluorescence drops dramatically. This inconsistency is likely due to human error during cell harvesting and preparation. Because microscopy indicates there were an unusual amount of crystals forming within 12.5uM of cisplatin slide, suggested cells may be left dried for an extended period of time, and proteins might be denatured. Interestingly, we expected cells to switch entirely to apoptotic pathways when we applied the maximum dosages of cisplatin to a cell culture (25 uM). However, we observed a significantly higher level of green immunofluorescence in Fig 1d, suggested its DNA repair pathway are the most active among all cisplatin treatments.
Cell cultures that were treated with UV light had a similar trend in response: when cells were exposed to 63 J/m2 of UV light, the level of PCNA expression are slightly lower than that of the control dish. As we increase the intensity of UV light to 125 J/m2 and 250 J/m2, the level of PCNA decreases accordingly, indicates that most of the cellular responses had switched to the apoptotic pathway. However, the result of the maximum dosages treatment (500 J/m2) were out of our expectation: like the maximum dosages cisplatin treatment, cells which endures maximum dosages of UV light exposure turn out to have the highest level of PCNA expression.
On the other hand, the result from immunoblotting is not consistent enough to be analyzed. By cross-comparison with other groups, we found out that no group observed any band from tubulin analysis, and there are heavy non-specific binding occurs on all cyclin D1 analysis. These outcomes indicated inappropriate secondary antibodies were selected for CD1/tubulin assay. However, as for γH2AX and geminin assay, we were misguided by the vague molecular marker; hence we cut on the incorrect side, therefore, there are only a few bands on the edge (fig d).
Previous investigations indicated: there are multiple cellular pathways which enables cells to perform DNA damage repair (Teruaki and David, 2013). Such a response is completed by an orchestration of protein activities, including some of the proteins we analyzed during this experiment, such as PCNA. Some previous studies stated that an increasing level of DNA damage leads to a rising PCNA expression (Balajee et al., 2001). These observations partially agrees with our experimental results. However, by applying a relatively higher dosage of damaging agents, we also realized that, after passing through a certain "threshold", the rise of genotoxicity could results in a gradual decrease in the level of PCNA, which we thought was due to a decrease in the amount of cells undergoes DNA repair and more cells undergoes apoptosis. This observation was also consistent with past findings, which indicates that PCNA activities can be maximally triggered under low levels of UV radiation(Soria et al., 2006).
However, none of any published reports observed sudden increase of PCNA expression when the level of DNA damage was raised from high levels to maximum dosages. There can be three possible explanations for such observations: the first and most likely explanation is: under low dosages, when DNA were repaired the expression of PCNA drop down to base level, whereas under maximum damage, cellular repair pathways are activated throughout the damaging process. The second possibility is: we made errors during experiment and cell cultures were stained differently. Last but not least, there might be a secondary threshold of DNA damage level that leads to acute cell cycle arrest followed by DNA repairing instead of cell apoptosis. Further studies needs to be conducted to testify our expected results on cyclin D1, geminin, tubulin and γH2AX.
To improve experimental design, we could devise more appropriate UV dosages to maintain the consistency of the experimental outcomes, because the result showed us UV light possess higher genotoxicity compared to cisplatin. We should also consider cell fates other than repair and apoptosis to post a more realistic hypothesis. Finally, a separate experiment to analyze the effect of time length of cell damaging to the cell responses, can be conducted to backup our theory.
As for experimental procedures, besides avoiding possible human errors, our experimental results could be more convincing if we were able to implement following improvements: firstly, we could split cells into at least 16 dishes. This improvement provides larger sample size and testing capacity. Secondly, microscopes with high magnification could be used to perform cell counting to analyze the level of cell apoptosis and cell arrest quantitatively, instead of speculating based on the level of indicators. Last but not least, a quantitative measurement for protein concentrations, such as UV absorption spectrum and iTRAQ technique, could be applied to produce a more reliable result.
Nowadays, we have made much progress toward the understanding of DNA damage responses. However, Some detailed mechanisms, for example, the regulation of protein activities in DDR and the exact magnitude of cell responses toward DNA damage, still remains mysterious to us. Although our experiment failed to unveil this mystery, many published experimental data do support our theories to some extent. Further investigations should be conducted to fully understand the orchestration of DDR mechanisms, and such knowledge will enable us to develop more effective clinical applications to treat DNA damage induced diseases.
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