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Quantification of Radiation Induced DNA Damage Response in Normal Skin Exposed in Clinical Settings
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
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. , p. 51
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 631
Keywords [en]
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: urn:nbn:se:uu:diva-134600ISBN: 978-91-554-7969-5 (print)OAI: oai:DiVA.org:uu-134600DiVA, id: diva2:373627
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
List of papers
1. Low-dose hypersensitive gammaH2AX response and infrequent apoptosis in epidermis from radiotherapy patients
Open this publication in new window or tab >>Low-dose hypersensitive gammaH2AX response and infrequent apoptosis in epidermis from radiotherapy patients
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2008 (English)In: Radiotherapy and Oncology, ISSN 0167-8140, E-ISSN 1879-0887, Vol. 88, no 3, p. 388-397Article 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.

Keywords
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:nbn:se:uu:diva-17482 (URN)10.1016/j.radonc.2008.04.017 (DOI)000260203800012 ()18524402 (PubMedID)
Available from: 2008-06-24 Created: 2008-06-24 Last updated: 2017-12-08Bibliographically approved
2. A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis
Open this publication in new window or tab >>A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis
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2010 (English)In: Radiotherapy and Oncology, ISSN 0167-8140, E-ISSN 1879-0887, Vol. 94, no 1, p. 90-101Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-134593 (URN)10.1016/j.radonc.2009.10.007 (DOI)000274889900014 ()19931928 (PubMedID)
Available from: 2010-11-29 Created: 2010-11-29 Last updated: 2017-12-12Bibliographically approved
3. 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
Open this publication in new window or tab >>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
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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:nbn:se:uu:diva-134594 (URN)
Available from: 2010-11-29 Created: 2010-11-29 Last updated: 2011-01-13
4. Mir-34a, mir-16 and mir-203 in the DNA damage response of epidermis and dermis to conventional radiotherapy
Open this publication in new window or tab >>Mir-34a, mir-16 and mir-203 in the DNA damage response of epidermis and dermis to conventional radiotherapy
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background:

The cellular response to radiation induced DNA damage determines the outcome of radiotherapy. The skin is a normal tissue that is always affected in radiotherapy, and exhibits both early and late effects that can be dose-limiting. However, the molecular mechanisms behind the side effects are still poorly understood The DNA-damage response pathways consist of a network of genes activated to repair DNA insult and promote cell survival. MicroRNAs (miRNAs) are short non-protein-coding RNAs that regulate ~30% of all gene expressions. Many miRNAs are tumor suppressors and activated by DNA damage. The role of miRNAs in the response to radiation induced DNA damage could be crucial and contribute to the different side effects seen in normal tissues.

Aim:

The objective of the present clinical study is to elucidate the potential impact of miRNA regulation of DNA damage response in normal skin to radiotherapy given with daily fractions of 2 Gy over 5 weeks. We have focused on miR-34a, miR-16 and MiR-203, known to be upregulated by p53 upon various types of genotoxic exposure.

Methods:

A total of 141 punch skin biopsies from 15 breast cancer patients were collected before, during and after radiotherapy. The skin was microdissected into epidermis and dermis. Micro-RNA levels were determined for miR-34a, miR-16 and miR-203 by RT-QPCR. Associated protein levels of p21, Bcl-2 and p63 were determined by immunohistochemistry.

Results:

miR-34a was induced in a very similar pattern as p21 during  the radiotherapy course. Also the decline rate of the miR-34a expression following the end of radiotherapy was equal to that of p21. The relative expression levels of miR-34a and p21 increased both in epidermis and dermis, to the same degree for the two different tissues during the treatment period (p< 0.002). After completion of treatment the miR-34a and p21 levels declined faster in the epidermis compared to dermis (p<0.003). The expression levels in dermis remained elevated for weeks for both miR-34a and p21. No change could be observed for miR-16 neither in epidermis or dermis, as well as in the expression of Bcl-2. Notably, no induced changes in miR-203 and p63 were detectable in epidermis. Unexpectedly, there was a significant reduction of miR-203 in the dermis.

Conclusion:

This study is the first to show a significant up-regulation of miR-34a in various normal cell populations to the DNA damage inflicted by radiotherapy. The miR-34a induction is of equal degree in epidermis and dermis and mirrors that of p21, a well-known target protein of p53. Secondly, in contrast to the successive decline of p21 and miR-34a in epidermal keratinocytes after completion of treatment, retention lasting for weeks is characteristic for fibroblasts and endothelial cells in dermis. This diverging molecular resolution of the DNA damage reveals early mechanistic differences in the pathological processes behind acute reversible effects of epidermis and late progressive changes of dermis. Our findings highlight that the accessibility makes the skin to a unique clinical model to quantify markers of DNA damage response to repeated DNA insult as it is delivered in radiotherapy.

 

National Category
Cancer and Oncology
Research subject
Oncology
Identifiers
urn:nbn:se:uu:diva-134597 (URN)
Available from: 2010-11-29 Created: 2010-11-29 Last updated: 2011-01-13

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