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Epidermal keratinocyte loss in response to daily 2 Gy fractions for 5 weeks of radiotherapy is associated with DSB-foci, growth arrest, apoptosis and lack of accelerated repopulation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background and purpose:

Tissue-sparing due to repopulation is expected to occur in epithelial cell populations during a course of fractionated radiotherapy (RT). Recently, we established, in the clinical setting of RT, the dose response relationship of subtherapeutic doses in terms of epidermal keratinocyte loss in the basal layer throughout 7 weeks of RT. Surprisingly, in the case of daily dose fractions of 1.10Gy, the keratinocyte loss per dose unit  increases over the last 4 weeks of the treatment period rather than being constant or decreasing. The aim of the present study is to elaborate on the issue of germinal keratinocyte response to daily dose fractions of 2.0Gy for 5 weeks. Here, we present assessments of keratinocyte loss, DSB foci, growth arrest, mitosis and apoptosis using methods earlier described by us.

Materials and Methods:

In total 240 skin punch biopsies, collected from 31 breast cancer patients, before, during and after postmastectomy radiotherapy given to the thoracic wall with daily 2.0Gy fractions for 5 weeks, were investigated. The dose response for basal keratinocyte density of the interfollicular epidermis was determined. The DNA damage response of keratinocytes was studied by immunostaining for molecular markers of DNA DSBs, growth arrest, mitosis and cell death using 53BP1, p21, phospho-H3 and γH2AX (apoptosis), respectively. The stainings of keratinocytes were counted manually or by digital image analysis.

Results:

The dose-response relationship for the loss of basal keratinocytes over 5 weeks of RT revealed a biphasic shape. An initial radioresistant phase was followed by an increase in radiosensitivity in the second part of RT. The rate of keratinocyte loss reflected the significant changes determined by 53BP1 and γH2AX foci 30 minutes after dose fractions over the treatment period. The highest induction of DSB foci per cell was observed towards the end of treatment. The increase in the fraction of p21 stained cells was also more prominent during the second half of the treatment as compared to the first period of RT. The apoptotic frequency was generally low but increased dramatically towards the end of RT. The mitotic cell number was significantly suppressed over 5 weeks, and did not recover during the weekend treatment-gaps. Notably, the mitotic rate increased more than threefold compared to unexposed skin, 2 weeks after the end of RT, followed by a rapid decline one week later.

Conclusion:

The dose response for germinal keratinocyte loss as a result of daily dose fractions of 2.0Gy over 5 weeks treatment deviates significantly from an exponential curve fit. The effectiveness of each dose fraction was less in the first half of the treatment when compared with the second half. No accelerated repopulation could be revealed over the 5 weeks, but was evident after completion of radiotherapy. The changes in keratinocyte response were associated with changes in induction of DSB foci and p21 protein expression, as well as apoptotic events over the treatment period.  In particular, we highlight the existence of pre-mitotic apoptosis, which increased significantly towards the end of 5 weeks RT. These findings suggest that it is necessary to reconsider the current conceptions regarding DNA repair, cell-cycle redistribution and repopulation of normal epithelial cells to a long-lasting courses of fractionated radiotherapy.

National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
URN: urn:nbn:se:uu:diva-134594OAI: oai:DiVA.org:uu-134594DiVA: diva2:372938
Available from: 2010-11-29 Created: 2010-11-29 Last updated: 2011-01-13
In thesis
1. 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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