A low-dose hypersensitive keratinocyte loss in response to fractionated radiotherapy is associated with growth arrest and apoptosis
Received 15 July 2009; received in revised form 5 October 2009; accepted 14 October 2009. published online 23 November 2009.
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 γH2AX 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 6.5weeks 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 γH2AX, 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 7weeks of radiotherapy (p<0.01). Growth arrest and cell proliferation assessed at 1week and 6.5weeks 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 6.5weeks 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 7weeks of radiotherapy. A persistent hypersensitive growth arrest response and cell killing mediate this effect.
ABSTRACT