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Cells exposed to ultraviolet (UV) rays of light often supress signalling pathways associated with inflammation as well as the immune system and as a result of activating the p38 mitogen activated protein kinase (MAPK) pathway. Radiation from UV light is from the electromagnetic spectrum and can be divided into two primary wavelengths; UVA 320 – 400 nm and UVB 290 – 320 nm. UVA rays are more dominant than UVB, however both UVA rays reaching the subcutaneous layer of skin and UVB rays reaching the dermis are known to cause the development of skin cancers via damaging the DNA of skin cells (1, 2, 3).
The effect of UV light on cells can be measured by determining the time the cells take to migrate back to their normal positions after the creation of an artificial wound. An inexpensive and easy assay to perform to make such measurements is the scratch assay. The scratch assay uses a pipette tip to create an artificial gap between the cells under aseptic conditions and uses high resolution imaging devices connected to an inverted microscope to take pictures of the cells migrating at specific time intervals. The images can then be used to calculate the distances travelled under the different UV exposures and therefore a comment on how the cells were affected under the different conditions can be made (1, 2, 3, 4).
In this experiment, Human squamous cell carcinoma (SSC) cells that are known to have a dysfunctional p53 proteins were taken from the epithelial Colo16 cell line and used to measure the migration distances of the cells under different UV exposures. Then the matrix metalloproteinase (MMP) inhibitor GM6001 was added to the Colo16 cells in order to determine if it had an effect on the cell migration distances under the same UV exposures (1, 3, 5, 6).
GM6001 is a MMP inhibitor that inhibits the enzyme collagenase which destroys the peptide bonds of the protein collagen found in the extracellular matrix. Collagen is an important component of the connective tissue and helps the cells adhere to their surrounding surfaces. GM6001 has been shown to block the phosphorylation of the epidermal growth factor receptors (EGFR) and inhibit cell migration responses (3, 5).
- To determine the migration rate of Colo16 cells exposed to UV light at intervals of 12 hours over 48 hours after the creation of an artificial wound (gap).
- To determine the effect of the MMP inhibitor GM6001 on the migration rate of Colo16 cells exposed to UV light at intervals of 24 hours over 72 hours after the creation of an artificial wound (gap).
If cells from the Colo16 cell line are treated with the MMP inhibitor GM6001 then it is expected that the mean gap distance will increase when compared with untreated Colo16 cells under the same exposure to UV light due to the inhibition of collagenases that breakdown collagen and the blocking of EGFR phosphorylation.
Materials and methods:
As per the BIOL2299 2014 Prac 4 lab notes.
Raw class data for the untreated and treated Colo16 cell gap distance (mm) is shown in Table 1 and Table 2 respectively.
From the class data our group was Group A shown in Table 3 and Table 4, and the mean class data along with its standard deviation values is shown in Table 5 to Table 8.
Table 1. Class Untreated Colo16 Cell Gap Distance (mm).
Table 2. Class Colo16 GM6001 Treated Cell Gap Distance (mm).
Table 3. Group A Colo16 Untreated Cell Gap Distance (mm).
Table 4. Group A GM6001 Treated Colo16 Cell Gap Distance (mm).
Table 5. Mean Class Colo16 Untreated Cell Gap Distance (mm).
Table 6. Class Standard Deviation (SD) of Colo16 Untreated Cell Gap Distance (mm).
Table 7. Mean Class GM6001 Treated Colo16 Cell Gap Distance (mm).
Table 8. Class Standard Deviation (SD) of GM6001 Treated Colo16 Cell Gap Distance (mm).
Figure 1. Line graph showing the mean gap distance of Untreated Colo16 cells under different UV exposures along with the standard deviation values after the creation of a synthetic gap using a pipette tip at intervals of 12 hours over 48 hours post-irradiation.
Figure 2. Line graph showing the mean gap distance of Colo16 cells treated with the MMP inhibitor GM6001 under different UV exposures along with the standard deviation values after the creation of a synthetic gap using a pipette tip at intervals of 24 hours over 72 hours post-irradiation.
From the line graph in Fig. 1, the Colo16 cells under UV radiation showed a slower decline in gap distance compared with the control. And the Colo16 cells exposed to UVA + UVB rays had the slowest decrease in gap distance over 48 hours after the creation of the artificial wound with a gap distance of only 0.200 mm suggesting that both UVA and UVB work with synergy together and the p38 MAPK pathway and therefore cause a delay in wound healing.
From the line graph in Fig. 2, the Colo16 cells treated with the MMP inhibitor GM6001 exposed to different UV conditions had a delayed cell migration response when compared to the control GM6001 Colo16 cells that were not exposed to UV irradiation. This may be due to the inhibition of collagenases that help breakdown the collagen found in the extracellular matrix (ECM) of the Colo16 cells via the action of GM6001.
GM6001 is a MMP inhibitor that inhibits the enzyme collagenase that normally is involved in the breakdown of collagen of the ECM. This inhibition may affect the cell migration of Colo16 cells during wound healing as a result of the collagen not being broken down by the collagenases in the ECM and therefore resulting in the cells being adhered to their surrounding for a longer time; increasing the time required to close the gap and complete the wound healing process.
- What other ways are there to determine whether cells are migrating into the wound or proliferating into it? (Are there morphological characteristics of cell migration or proliferation?)
Other ways of determining cell migration or proliferation include:
1 – Immunofluorescence staining: cells can be stained using fluorescence markers that use antibodies to bind to specific antigens associated with cell migration or proliferation such as the proliferating cell nuclear antigen (PCNA) (7).
2 – Laser scanning confocal microscopy: cells can be viewed under high resolution and the morphological features of migrating cells can be observed such as the formation of fibrin matrices, an increase of length, width and total spread of fibroblasts (7).
3 – Boyden chamber: which measures cell migration via the determination of a chemical gradient based on chemicals release via chemotaxis during cell migration (7).
- What is a Boyden chamber and why would you use it? Give 3 examples of where it can be used.
A Boyden chamber is an in vitro chemotaxis assay that measures cell migration via the analysis of leukocyte chemotaxis, where cells are placed in pores at the top of a chamber and allowed to migrate through its pores containing chemotactic agents. After incubation the cells are stained and the number of cells that have migrated to the lower portion of the chamber (8).
The Boyden chamber can be used to measure:
1 – Cell migration based on chemicals released to the cell’s surrounding, and determine if a specific chemical causes the cells to migrate towards or away from the stimulant (Chemotaxis) (8).
2 – Cell migration based on the gradient of specific extracellular matrix proteins, and determine if a particular protein causes the cells to migrate towards or away from the protein. This can be done via the coating of the chamber with the protein of choice (Haptotaxis) (8).
3 – Cell migration rate of tumour cells through the vascular endothelium toward specific chemokines (8).
- Describe 3 limitations of the scratch method.
Some limitations of the scratch method include:
1 – Creation of an artificial wound of the same approximate width can be extremely difficult and any variance in the gap distance between the different cells at time 0 hr can greatly affect the significance of the results (4).
2 – The incubation of the cells after the creation of the wound (scratch) has to be long enough to help the cells migrate during the fastest time frame and any variance in this may lead to the cells not migrating equally, therefore affecting the results. Also, the temperature and environmental conditions of the incubator have to be at their ideal values for healthy cellular migration (4).
3 – Highly time consuming, in order for the readings to be statistically significant at least 100 readings of distance for each sample and each experiment must be repeated at least three times. As a result, this creates copious amounts of data that consumes a lot of time and energy to analyse (4).
- If you are investigating cell growth factors on cell migration, should serum be present in the media? Explain your answer.
Yes, Serum containing the growth factors should be found in the media in order to study its effects and the growth factors should be soluble in the serum and added to the media before the addition of cells in order to be equally distributed in the solution (4).
- What are the main differences in investigating a scratch assay using non-transformed vs transformed cells?
Transformed cells are transfected with a plasmid coding the gene of interest along with a plasmid marker before the conduction of a scratch assay, whereas non-transformed cells are investigated without being transfected with a specific plasmid (4).
- Apart from taking photos of the cells over time, propose another way you may be able to show cell migration.
Cell migration can be shown via the conduction of an electric cell-substrate impedance sensing (ECIS) in vitro cell migration assay that measures cell migration via the creation of a wound by passing an electrical current through the cell and causing electroporation. Then cell migration is measured via the calculation of the cell’s impedance in ohms over a specific time as seen in Fig. 3 (9).
Figure 3. Typical ECIS data involving the electric cell-substrate impedance sensing (ECIS) in vitro cell migration assay (http://www.biophysics.com/woundhealingpubs.php#ECIStheory).
- Which of the methods is the one that most labs would not use, explain why you think this would be so.
Most labs would not use the ECIS in vitro migration assay due to its expensive cost and it’s relatively time consuming cell type dependant incubation times. Another in vitro migration assay that would not be used in labs is the microfluidics-based system due to its need for nanofabrication facilities and its reasonably very expensive cost (4).
In conclusion, Colo16 cells under UV radiation showed a slower decline in gap distance compared with the control. And the Colo16 cells exposed to UVA + UVB rays had the slowest decrease in gap distance over 48 hours after the creation of the artificial wound with a gap distance of only 0.200 mm suggesting that both UVA and UVB work with synergy together and the p38 MAPK pathway and therefore cause a delay in wound healing.
Whereas, the Colo16 cells treated with the MMP inhibitor GM6001 exposed to different UV conditions had a delayed cell migration response when compared to the control GM6001 Colo16 cells that were not exposed to UV irradiation. This may be due to the inhibition of collagenases that help breakdown the collagen found in the extracellular matrix (ECM) of the Colo16 cells via the action of GM6001.
This inhibition caused by GM6001 may affect the cell migration of Colo16 cells during wound healing as a result of the collagen not being broken down by the collagenases in the ECM and therefore resulting in the cells being adhered to their surrounding for a longer time; increasing the time required to close the gap and complete the wound healing process.
- BIOL2299 Biology of Tissue Growth and Repair Manual, 2014.
- Reichrath J, Rass K. Ultraviolet damage, DNA repair and vitamin D in nonmelanoma skin cancer and in malignant melanoma: an update. Advances in experimental medicine and biology. 2014;810:208-33.
- Muthusamy V, Piva TJ. A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines. Archives of dermatological research. 2013;305(9):817-33.
- Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols. 2007;2(2):329-33.
- Grobelny D, Poncz L, Galardy RE. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry. 1992;31(31):7152-4.
- Moore GE, Merrick SB, Woods LK, Arabasz NM. A Human Squamous Cell Carcinoma Cell Line. Cancer Research. 1975;35(10):2684-8.
- Chen HC. Boyden chamber assay. Methods in molecular biology (Clifton, NJ). 2005;294:15-22.
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