| 1. |
- Fessé, Per, et al.
(författare)
-
UV-Radiation Response Proteins Reveal Undifferentiated Cutaneous Interfollicular Melanocytes with Hyperradiosensitivity to Differentiation at 0.05 Gy Radiotherapy Dose Fractions.
- 2019
-
Ingår i: Radiation research. - 1938-5404 .- 0033-7587. ; 191:1, s. 93-106
-
Tidskriftsartikel (refereegranskat)abstract
- To date, the response activated in melanocytes by repeated genotoxic insults from radiotherapy has not been explored. We hypothesized that the molecular pathways involved in the response of melanocytes to ionizing radiation and ultraviolet radiation (UVR) are similar. Skin punch biopsies, not sun-exposed, were collected from prostate cancer patients before, as well as at 1 and 6.5 weeks after daily doses of 0.05-1.1 Gy. Interfollicular melanocytes were identified by ΔNp63- and eosin-periodic acid Schiff staining. Immunohistochemistry and immunofluorescence were performed to detect molecular markers of the melanocyte lineage. Melanocytes were negative for ΔNp63, and the number remained unchanged over the treatment period. At radiation doses as low as 0.05 Gy, melanocytes express higher protein levels of microphthalmia-associated transcription factor (MITF) and Bcl-2. Subsets of MITF- and Bcl-2-negative melanocytes were identified among interfollicular melanocytes in unexposed skin; the cell number in both subsets was reduced after irradiation in a way that indicates low-dose hyperradiosensitivity. A corresponding increase in MITF- and Bcl-2-positive cells was observed. PAX3 and SOX10 co-localized to some extent with MITF in unexposed skin, more so than after radiotherapy. Low doses of ionizing radiation also intensified c-KIT and DCT staining. Nuclear p53 and p21 were undetectable in melanocytes. Apoptosis and proliferation could not be observed. In conclusion, undifferentiated interfollicular melanocytes were identified, and responded with differentiation in a hypersensitive manner at 0.05 Gy doses. Radioresistance regarding cell death was maintained up to fractionated doses of 1.1 Gy, applied for 7 weeks. The results suggest that the initial steps of melanin synthesis are common to ionizing radiation and UVR, and underline the importance of keratinocyte-melanocyte interaction behind hyperpigmentation and depigmentation to radiotherapy.
|
|
| 2. |
- Qvarnström, Fredrik, et al.
(författare)
-
Double strand break induction and kinetics indicate preserved hypersensitivity in keratinocytes to subtherapeutic doses for 7weeks of radiotherapy
- 2017
-
Ingår i: Radiotherapy and Oncology. - : Elsevier BV. - 0167-8140 .- 1879-0887. ; 122:1, s. 163-169
-
Tidskriftsartikel (refereegranskat)abstract
- Background and purpose Previously we reported that hyper-radiosensitivity (HRS) was evidenced by quantifying DNA double strand break (DSB) foci in epidermis biopsies collected after delivering radiotherapeutic one and five dose fractions. The aim of this study was to determine whether HRS was preserved throughout a 7-week radiotherapy treatment, and also to examine the rate of foci decline and foci persistence between dose fractions. Materials and methods 42 patients with prostate cancer received 7-week fractionated radiotherapy treatment (RT) with daily dose fractions of 0.05–1.10Gy to the skin. Before RT, and at several times throughout treatment, skin biopsies (n=452) were collected at 30min, and 2, 3, 24, and 72h after dose fractions. DSB-foci markers, γH2AX and 53BP1, were labelled in epidermal keratinocytes with immunofluorescence and immunohistochemical staining. Foci were counted both with digital image analysis and manually. Results HRS in keratinocytes was evidenced by the dose–response relationships of DSB foci, observed throughout the treatment course, independent of sampling time and quantification method. Foci observed at 24h after dose fractions indicated considerable DSB persistence. Accordingly, foci significantly accumulated after 5 consecutive dose fractions. For doses below 0.3Gy, persistent foci could be observed even at 72h after damage induction. A comparison of γH2AX and 53BP1 quantifications in double-stained biopsies showed similar HRS dose–response relationships. Conclusions These results represented the first evidence of preserved HRS, assessed by γH2AX- and 53BP1-labelled DSB foci, throughout a 7-week treatment course with daily repeated subtherapeutic dose fractions. © 2016 Elsevier Ireland Ltd
|
|
| 3. |
- Qvarnström, Olov Fredrik, et al.
(författare)
-
DNA double strand break quantification in skin biopsies.
- 2004
-
Ingår i: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology. - : Elsevier BV. - 0167-8140. ; 72:3, s. 311-7
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND PURPOSE: Following induction of double strand breaks the histone H2AX is rapidly phosphorylated at regions flanking the breaks resulting in nuclear gamma H2AX foci. The purpose of this study was to use this endogenous signalling system to quantify the in vivo response to radiation in normal tissue. PATIENTS AND METHODS: Skin biopsies were taken from prostate cancer patients undergoing radiotherapy with a curative intent. Biopsies were taken at locations corresponding to 5 different doses in the range below 1.1 Gy per fraction. Biopsies were taken from patients 30 min following the first fraction and then once again following the fraction given after the first weekend break in the treatment course. The DNA double strand breaks were visualised as gamma H2AX foci using immunohistochemistry. Images were acquired using a CCD-camera and a fluorescence microscope and the gamma H2AX foci were quantified using digital image analysis including the basic procedures of top-hat transformation, threshold setting and labelling. RESULTS: Repeated assessments of the biopsies showed a high reproducibility in quantifying the number of foci per DNA area of the nucleated cells in epidermis. The reproducibility was equally good for the two biopsy occasions. A linear dose response was observed for the epidermis in the dose region 0-1 Gy. CONCLUSIONS: We have established a method to measure the relative amount of DNA double strand breaks by detecting gamma H2AX foci in patients exposed to radiotherapy. The method provides a tool to study induction and repair of DNA double strand breaks and has the potential to predict individual radiosensitivity.
|
|
| 4. |
- Simonsson, Martin, et al.
(författare)
-
Low-dose hypersensitive gammaH2AX response and infrequent apoptosis in epidermis from radiotherapy patients.
- 2008
-
Ingår i: Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology. - : Elsevier BV. - 0167-8140 .- 1879-0887. ; 88:3, s. 388-97
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND PURPOSE: A low-dose hypersensitivity to radiation can be observed in vitro for many human cell types in terms of increased cell kill per dose unit for doses below 0.5Gy. Quantification of the double-strand break marker gammaH2AX in samples taken in clinical radiotherapy practice has the potential to provide important information about how induction and repair of severe DNA damage and apoptosis are linked to low-dose hypersensitivity. MATERIAL AND METHODS: The effects of exposure to low doses (0.05-1.1Gy) were investigated in skin biopsies taken from prostate cancer patients undergoing the first week of radiotherapy. gammaH2AX foci and apoptotic cells were visualised by immunohistochemistry and quantified by image analysis. RESULTS: The gammaH2AX foci pattern in biopsies taken 30min after a single fraction revealed a low-dose hypersensitivity below 0.3Gy (p=0.0009). The result was consistent for repeated fractions (p=0.00001). No decrease in foci numbers could be detected when comparing biopsies taken 30min and 2h after single fractions of 0.4 and 1.2Gy. The result was consistent for repeated fractions. Only 43 of 168,000 cells in total were identified as apoptotic, yet a dose dependency could be detected after 1week of radiotherapy (p=0.003). CONCLUSIONS: We describe a method based on gammaH2AX to study DNA damage response and apoptosis in a clinical setting. A gammaH2AX hypersensitive response to low doses can be observed in epidermal skin, already 30min following delivered fraction. A very low frequency of apoptosis in normal epithelium suggests that this effect is not an important part of the in vivo response to low doses.
|
|
| 5. |
- Turesson, Ingela, et al.
(författare)
-
A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis.
- 2010
-
Ingår i: Radiotherapy and oncology. - : Elsevier BV. - 1879-0887 .- 0167-8140. ; 94:1, s. 90-101
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND AND PURPOSE: The existence of a hypersensitive radiation response to doses below 0.5Gy is well established for many normal and tumour cell lines. There is also evidence for hypersensitive tissue responses in acute skin damage and kidney function in mice. Recently, we have identified that a hypersensitive gammaH2AX response exists in human epidermis. The aim of this study was to investigate the dose-response of basal clonogenic keratinocytes in normal skin to fractionated radiotherapy with low dose fractions. MATERIALS: Skin punch biopsies were taken before and during radiotherapy from prostate cancer patients undergoing radiotherapy with a curative intent. Areas of epidermis receiving daily fractions of approximately 0.1, 0.2, 0.45 and 1.1Gy were biopsied on the same occasion to determine dose-response for each individual patient. In total, 89 cases were assessed either at 1, 2.5, 3, 4, 5 or 6.5 weeks in the treatment course. Biopsy sampling of another 25 patients was performed from areas receiving 0.45 and 1.1Gy per fraction at regular intervals throughout the 7-week treatment period. The number of basal keratinocytes per mm of the interfollicular epidermis was determined. The DNA damage response of the basal keratinocytes was investigated by immunohistochemical staining for molecular markers of growth arrest, mitosis and cell death, using p21, phospho-H3 and gammaH2AX, respectively. The number of stained keratinocytes in the basal layer was counted manually. The p21 staining was also quantified by digital image analysis. RESULTS: The individual dose-response relationships revealed a low-dose hypersensitivity for reduction of basal keratinocytes throughout 7 weeks of radiotherapy (p<0.01). Growth arrest and cell proliferation assessed at 1 week and 6.5 weeks showed, in both cases, hypersensitive increase of p21 (p<0.01) and hypersensitive depression of mitosis (p<0.01). Manual counting and digital image analysis of p21 showed good agreement. Cell death was infrequent but increased significantly between 1 and 6.5 weeks and displayed a hypersensitive dose-response at the end of the treatment period. CONCLUSIONS: A low-dose hypersensitivity in basal skin keratinocyte reduction is present throughout 7 weeks of radiotherapy. A persistent hypersensitive growth arrest response and cell killing mediate this effect.
|
|
