http://www.liebertonline.com/doi/pdfplus/10.1089/ten.2006.12.3395
Quantitative Analysis of Radiation-Induced DNA Break Repair
in a Cultured Oral Mucosal Model
HINNE A. RAKHORST, M.D.,1 WENDY M.W. TRA, B.Sc.,1 SANDRA T.
POSTHUMUS-VAN SLUIJS, B.Sc.,1 STEVEN E.R. HOVIUS, M.D., Ph.D.,1 PETER C.
LEVENDAG, M.D., Ph.D.,2 ROLAND KANAAR, Ph.D.,2,3 and STEFAN O.P. HOFER, M.D., Ph.D.1
This study uses tissue engineering to study the effects of radiation on skin tissue from the mucosal membrane. This work is extremely relevant to the condition of oral mucositis, a side effect experienced by many patients receiving chemotherapy. This condition can consist of ulcers in the mouth and even malnutrition. Usually this condition is treated by stopping the chemotherapy until the skin heals, which unfortunately allows for the spread of cancer. Using oral keratinocytes and fibroblasts and, experiments were conducted in vitro to assess the effects of radiation on the tissue, especially its effect on proteins involved with the DNA double-strand break repair mechanism. I feel that this paper is both interesting and important because it showcases how tissue engineering can be used to create a very important in vitro model to test conditions that can be dangerous or unfeasible in vivo.
The first step to the experiment involved constructing a mucosal substitute. This was done by obtaining fibroblasts from human buccal mucosa. Fibroblasts and keratinocytes were seeded into an acellular dermal carrier and were then gamma-irradiated. Next, the tissue cultures were examined to quantify P53 binding protein 1, MRE11 and RAD51—all proteins involved in double strand break (DSB) repair. These proteins were quantified using antibodies; furthermore, live-dead assays were performed to measure cell proliferation and apoptosis due to radiation.
The results indicated clear changes in the cell and tissue morphology as a result of the radiation. First the nuclei became much smaller and showed chromatin condensation (pyknosis). Further staining showed increased apoptosis most likely due to extreme damage to the DNA. The amount of the DSB-repair proteins was quantified through immunofluorescence visualization. These DSB-repair proteins would conglomerate around the site of the DSB forming ionizing radiation-induced foci (IRIF) in the nucleus. These IRIFs could be visualized, and thus the number of IRIFs per nucleus could be calculated and measured as an indication of the amount of double strand breaks that were occurring due to radiation. Using the protein P53, the expected trend of increased IRIFs with increased radiation occurred. However, in the case of other proteins such as MRE11, the pattern was less clear; thus the MRE11 protein is not as good of an indicator of double strand break levels. Cell proliferation staining also did show some peaks despite radiation at the low 2 Gy level indicating the potential for the cells to recuperate and grow. Overall, this study was a good model to display the effect of radiation on DNA double strand breakage; further study of the radiation effects with full understanding of cell cycle and immunological responses (i.e. cytokines), could lead to an even better understanding of radiation on skin tissue and how this may affect oral mucositis.
9 comments:
From my understanding of the paper, they aimed to use tissue engineering as a means to actually test the effects of radiation on the tissue or basically to create an in-vitro model--since actually using patients was not a realistic option. In their work, their goal was actually to use tissue engineering to mimic mucosa tissue/create a realistic model fo r testing. They actually collected human skin, removed the epidermis, and took just the dermis and acellularized it (by gamma radiation). Then they were able to seed this acellular dermis with mucosal fibroblasts and keratinocytes. I think the authors saw this paper as a first step in tissue engineering to provide more quantitative data in analyzing results to help in clinical studies and that future results from experiments with tissue engineered models could aid in discovering treatments for mucositis. As far as I read, they were not actually attempting to re-create mucosal tissue for implantation into patients sufforing from mucositis.
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*suffering
sorry! :P
What is the reason that they chose mucosal tissues for their experimentation?
Is it because mucosal tissues are most sensitive to radiation in treating cancer? Do mucosal tissues necessarily represent risks of radiation oncology?
The aim of the article is to study oral mucositis which can be a painful side effect of ulcers in the mucosa tissue experienced by cancer patients being treated with chemotherapy. The reason why mucosa tissue is so susceptible to radiation is that compared to other tissues it has a "physiologically high turnover rate," and because radiation targets highly prolifative cells the most (i.e. tumors), the mucosa tissue is also more affected than other tissues.
What were the time points utilized for immunostaining analysis? In addition, you mentioned markers like p53 which is an apoptotic marker as well. Did the group do any experimental analysis utilizing repair mechanisms... like 53BP1 g-H2AX or Ku80(specifically for dsDNA breaks)? Also, 2 Gy is a significant dosage in radiation biology. For instance, if that amount of radiation is given in an acute dosage to the eye, cataracts will form.
53BP1 is the P53 binding protein 1, which they tested for. As mentioned, MRE11 (bridges broken DNA ends) and RAD51 (promotes pairing of intact identical sequences on sister chromatid) are also both DSB repair proteins which were tested for. No mention of Ku80 was made.
As for the radiation levels, 2+ Gy radiation levels are fairly standard across studies, and this also appears to be a radiation level that is practically used in chemotherapy as well (which is what the study was trying to mimic).
Did the paper only use gamma radiation to test the effects on mucosal tissue? It would be interesting to see if any other types of radiation would have an effect on these tissues, namely UV radiation. (like our project) haha =). So gamma radiation was chosen just because it is part of chemotherapy?
Yup. No UV radiation testing was conducted in this study, just gamma radiation.
wow... I didn't know that 2 Gy was still a preferred dosage these days for radiotherapy. Is it normally split up to discourage the amount of healthy tissue death/is the dosage specific for skin? I know that cancer cells are normally hypoxic so they are more resistant to radiation as they don't form as many free radicals. Cancer... ahhh Potter St. love.
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