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Low-dose hypersensitive gammaH2AX response and infrequent apoptosis in epidermis from radiotherapy patients
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. (Prostata)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology. (Prostata)
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2008 (English)In: Radiotherapy and Oncology, ISSN 0167-8140, E-ISSN 1879-0887, Vol. 88, no 3, 388-397 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2008. Vol. 88, no 3, 388-397 p.
Keyword [en]
γH2AX, Hypersensitivity, Apoptosis, Epidermis, Normal tissue, Parp-1; DNA damage, DNA repair, Double strand breaks, DSB, Digital image analysis, Foci
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-17482DOI: 10.1016/j.radonc.2008.04.017ISI: 000260203800012PubMedID: 18524402OAI: oai:DiVA.org:uu-17482DiVA: diva2:45253
Available from: 2008-06-24 Created: 2008-06-24 Last updated: 2017-12-08Bibliographically approved
In thesis
1. DNA Damage Response of Normal Epidermis in the Clinical Setting of Fractionated Radiotherapy: Evidence of a preserved low-dose hypersensitivity response
Open this publication in new window or tab >>DNA Damage Response of Normal Epidermis in the Clinical Setting of Fractionated Radiotherapy: Evidence of a preserved low-dose hypersensitivity response
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Investigations of DNA damage response (DDR) mechanisms in normal tissues have implications for both cancer prevention and treatments. The accumulating knowledge about protein function and molecular markers makes it possible to directly trace and interpret cellular DDR in a tissue context.

Using immunohistochemical techniques and digital image analysis, we have examined several principal DDR events in epidermis from patients undergoing fractionated radiotherapy. Acquiring biopsies from different regions of the skin provides the possibility to determine in vivo dose response at clinically relevant dose levels throughout the treatment.

A crucial event in cellular DDR is the repair of DNA double strand breaks (DSBs). These serious lesions can be directly visualised in cells by detecting foci forming markers such as γH2AX and 53BP1. Our results reveal that DSB-signalling foci can be detected and quantified in paraffin-embedded tissues. More importantly, epidermal DSB foci dose response reveals hypersensitivity, detected as elevated foci levels per dose unit, for doses below ~0.3Gy. The low-dose hypersensitive dose response is observed throughout the treatment course and also in between fractions: at 30 minutes, 3 hours and 24 hours following delivered fractions. The dose response at 24 hours further reveals that foci levels do not return to background levels between fractions. Furthermore, a low-dose hypersensitive dose response is also observed for these persistent foci.

Investigations of end points further downstream in the DDR pathways confirmed that the low-dose hypersensitivity was preserved for: the checkpoint regulating p21 kinase inhibitor; mitosis suppression; apoptosis induction and basal keratinocyte reduction.

Our results reveal preserved low-dose hypersensitivity both early and late in the DDR pathways. A possible link between the dose-response relationships is therefore suggested. The preserved low-dose hypersensitivity is a cause for re-evaluation of the risks associated with low-dose exposure and has implications for cancer treatments, diagnostics and radiation protection.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 457
Series
Keyword
DNA damage response, low-dose hypersensitivity, dose response, normal tissue, epidermis, keratinocyte, fractionated radiotherapy, DNA double strand break, DSB, foci, γH2AX, 53BP1, checkpoint, p21, apoptosis, mitosis
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:uu:diva-101075 (URN)978-91-554-7523-9 (ISBN)
Public defence
2009-06-03, Universitetshuset sal IX, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-05-13 Created: 2009-04-17 Last updated: 2009-05-13Bibliographically approved
2. Quantification of Radiation Induced DNA Damage Response in Normal Skin Exposed in Clinical Settings
Open this publication in new window or tab >>Quantification of Radiation Induced DNA Damage Response in Normal Skin Exposed in Clinical Settings
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The structure, function and accessibility of epidermal skin provide aunique opportunity to study the DNA damage response (DDR) of a normaltissue. The in vivo response can be examined in detail, at a molecularlevel, and further associated to the structural changes, observed at atissue level. We collected an extensive skin biopsy material frompatients undergoing fractionated radiotherapy for 5 to 7 weeks. Several end-points inthe DDR pathways were examined before, during and after the treatment.

Quantification of DNA double strand break (DSB) signalling focirevealed a hypersensitivity to doses below 0.3Gy. Furthermore, aconsiderable amount of foci persisted between fractions. The low dosehypersensitivity was observed throughout the treatment and was alsoobserved for several key parameters further downstream in the DDR-pathway, such as p21-associated checkpoint activation, apoptosisinduction and reduction in basal keratinocyte density (BKD).Furthermore, for dose fractions above 1.0 Gy, a distinct acceleration inDDR was observed half way into treatment. This was manifested as anaccelerated loss of basal keratinocytes, mirrored by a simultaneousincrease in DSBs and p21 expression.

Quantifications of mitotic events revealed a pronounced suppression ofmitosis throughout the treatment which was clearly low dosehypersensitive. Thus, no evidence of accelerated repopulation could beobserved for fraction doses ranging from 0.05 to 2Gy.

Our results suggest that the keratinocyte response primarily isdetermined by checkpoints, which leads to pre-mitotic cell elimination by permanent growth arrest and apoptosis.

A comparison between the epidermal and dermal sub-compartments revealsa consistent up-regulation of the DDR response during treatment. Adifference was however observed in the recovery phase after treatment,where miR-34a and p21 remain up-regulated in dermis more persistentlythan in epidermis. Our observations suggest that the recovery phaseafter treatment can provide important clues to understand clinicalobservations such as the early and late effects observed in normaltissues during fractionated radiotherapy.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 51 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 631
Keyword
DNA damage response, low-dose hypersensitivity, dose response, normal tissue, epidermis, dermis, keratinocyte, fractionated radiotherapy, DNA double strand break, DSB, foci, gamma-H2AX, 53BP1, p21, checkpoint, apoptosis, mitosis, micro-RNA, miR-34a
National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:uu:diva-134600 (URN)978-91-554-7969-5 (ISBN)
Public defence
2011-01-14, Hörsal Betty Pettersson, Blåsenhus, von Kraemers Allé 1, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2010-12-23 Created: 2010-11-29 Last updated: 2011-01-13Bibliographically approved

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Simonsson, MartinQvarnström, FredrikTuresson, Ingela

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