Patients and Radiotherapy

Skin biopsies were taken from 33 prostate cancer patients receiving radiotherapy with curative intent in Gothenburg, Sweden, between 2003 and 2005. The median age of the patients was 65 years (range 49–74 years). Approval was obtained from the Ethical Committee at the University of Gothenburg (Gothenburg, Sweden). Written informed consent was obtained from all patients prior to participation.

In accordance with ICRU50 (34), the prescribed radiation dose to the isocenter of the prostate was 35 daily fractions of 2 Gy for 7 weeks. Photons (11 or 15 MV) were applied in a three-field technique with one anterior field and opposed lateral fields with wedges. A 5-mm bolus was added on the left lateral field. The dose per fraction, at a depth of 0.1 mm below the skin surface (relevant for the biological end points), was determined carefully at the location where each biopsy was taken, as described in detail earlier (3, 4).

A total of 268 skin punch biopsies, 3 mm in diameter, were sampled. Four unexposed control biopsies (0 Gy) were collected from the hip region, not sun-exposed, for each patient prior to the start of the radiotherapy course and before the CT for dose planning and the simulation procedure. Four irradiated biopsies were taken during radiotherapy from each patient at the same occasion from skin areas that would correspond to doses of approximately 0.1, 0.2, 0.45 and 1.1 Gy, respectively. The two lowest dose per fractions were obtained by taking biopsies at 15 and 30 mm outside the lateral fields in the penumbra region. The two highest dose per fractions were obtained by taking biopsies within the lateral fields. The position of each sample in relationship to the treatment fields was transferred to a transparency film and was compared to the dose plan to further improve the dose determination for each of the four irradiated biopsies. The doses in the build-up region both inside and outside the treatment fields were determined by ionization chamber measurements in phantoms with a typical field set-up for these patients, as described in detail elsewhere (3). The estimated maximum uncertainty of the determined doses was less than 12% for the two lower doses and less than 7% for the two higher doses. The unique biopsy sampling procedure made it possible to determine dose-response relationships for each individual patient and all investigated end points for the cells of the melanocyte lineage.

The average and standard deviation of the individual dose estimations for each biopsy taken from the four different sites for all 33 patients were 0.05 ± 0.01, 0.13 ± 0.05, 0.44 ± 0.03 and 1.09 ± 0.08 Gy. Biopsies from the 33 patients were collected at two occasions during the radiotherapy course: group 1 (22 patients) at 1 week of radiotherapy and group 2 (12 patients) 6.5–7 weeks of radiotherapy. All biopsies were taken 30 min after the latest fraction and fixed immediately in 4% formaldehyde, dehydrated and embedded in paraffin.

Immunohistochemistry

Three transverse 4-µm tissue sections from each biopsy were taken from various levels and mounted on a Superfrost^™ Plus Slide (Menzel-Gläser, Braunschweig, Germany). The slides were dried at 37°C overnight. Immunohistochemical staining was performed on a Ventana Benchmark^® automated IHC stainer using the Ventana iView^™ DAB detection kit (Ventana^® Medical Systems, Tucson, AZ); subsequent manual counterstaining was performed with Meyers HTX. The primary antibodies were: monoclonal mouse antibody ΔNp63 (4A4) sc-8431, raised against amino acids 1-205 at the N-terminus of human ΔNp63 (1:200; Santa Cruz Biotechnology^® Inc., Dallas, TX); monoclonal mouse antibody MITF clone D5 (1:50; Dako, Glostrup, Denmark); and monoclonal mouse antibody Bcl-2 clone 124 (1:15; Dako). In addition, three tissue sections from each biopsy were stained with hematoxylin and eosin (H& E) and periodic acid-Schiff (eosin-PAS). Tissues known to express the antigen of interest were used as positive controls. As negative controls, skin biopsy sections omitting the primary antibodies from the staining procedure were used. For each molecular marker, all tissue sections from one patient were stained simultaneously to avoid influence from fluctuations in the procedure.

Immunofluorescence

Combinations of primary antibodies from different species were used for double-staining: MITF (1:100, monoclonal mouse, clone D5; Dako), MITF (1:50, polyclonal rabbit; Atlas Antibodies, Bromma, Sweden), ΔNp63 (1:200, monoclonal mouse, clone 4A4; Santa Cruz Biotechnology), ΔNp63 (1:100, polyclonal rabbit; Atlas Antibodies), Bcl-2 (1:20, monoclonal mouse, clone 124; Dako), p53 (1:100, monoclonal mouse, clone DO-7; Dako), Ki-67 (1:100, monoclonal mouse, clone MIB-1; Dako), SOX10 (1:100, polyclonal goat, N-20; Santa Cruz Biotechnology), PAX3 (1:100, polyclonal rabbit; Invitrogen), p21 (1:100, monoclonal mouse, clone EA10; Abcam^®, Cambridge, MA), c-KIT (1:100, polyclonal rabbit; Dako) and DCT (1:500, monoclonal mouse, clone C-9; Santa Cruz Biotechnology). Double-staining experiments were performed with appropriate pairs of fluorescent secondary antibodies raised in goat or donkey and attached to Alexa Fluor^® 480 and 555 fluorescent dyes (1:100; Molecular Probes^®, Eugene, OR). The manual staining protocol included epitope retrieval in boric acid buffer (pH 7.0) heated in a water bath (90°C) for 45 min. Antibody incubations were performed at 20°C for 1 h and followed by three 5-min washes in phosphate-buffered saline (pH 7.4). 4′,6-diamidino-2-phenylindole dilactate (DAPI, 0.4 µg/ml; Molecular Probes) was used for nuclear staining. Slides with air-dried sections were mounted in Vectashield^® mounting media (Vector^® Laboratories, Burlingame, CA).

Quantification of Molecular Markers

Quantitative estimates of melanocytes positive or negative for ΔNp63, MITF and/or Bcl-2 were determined by cell counting restricted to the basal layer of the epidermis. These cells can easily be distinguished from keratinocytes through the morphological characteristics revealed by eosin-PAS staining (5, 6, 35); melanocytes have distinct nucleoli, tendency for vacuolization, cytoplasm adherence to the nucleus and lack of desmosomes. ΔNp63 is a cell-cycle regulator expressed in keratinocytes but undetectable in normal melanocytes (36). The ΔNp63-negative cells fulfilled the morphological criteria of melanocytes. Therefore, all ΔNp63-negative cells were associated with the melanocyte lineage. The cell nucleus of the melanocyte very distinctly stains for MITF. In contrast, Bcl-2 protein is observed in the cytoplasm of melanocytes. However, Bcl-2 expression is undetectable in keratinocytes in the unexposed epidermis and also upon UVR exposure (37).

The total number of cells in the melanocyte lineage identified per mm of basal membrane was determined for all biopsies on three separate sections for eosin-PAS staining and each immunostaining. All counting was performed by one of the authors (P.F.) using a brightfield microscope with a 100× objective. The author I.H. also counted all eosin-PAS samples, with results equivalent to those of PF (linear regression, R=0.84, P < 0.001, data not shown). With the use of 100× magnification the desmosomes of the keratinocyte cell membrane were clearly visualized in all immunohistochemical staining, facilitating the separation of melanocytes from keratinocytes located at the basement membrane.

TABLE 1

Mean Number of Cells per mm for each Marker and Patient Group

To evaluate the presence of melanocyte-specific markers and DNA damage proteins expressed in melanocytes, ΔNp63 co-staining was assessed by immunofluorescence. We identified subpopulations positive or negative for each protein and co-expression of melanocyte-specific markers. An estimation of the number of cells within each subset of markers was intended.

Statistical Analysis

Repeated measures analysis of variance (ANOVA) was used for comparisons among the two patient groups. The correlation between dose and response for each individual was determined by Kendall's tau for each staining (38). The mean values of the individual correlation coefficients were tested in a one-sample t test with the hypothesis that the true mean equals zero. Statistical Package for the Social Sciences (SPSS version 22; Chicago, IL) and R were used for all statistical analyses (39).

Low-dose hypersensitivity was investigated by assessing the shapes of the individual dose-response curves for estimates of MITF- and Bcl-2-negative melanocytes. Individual dose-response relationships were normalized to dose fraction, providing a measure by which the effect per dose unit is comparable within each patient's biopsy set. The slopes from all individual regressions were collected and tested using a t test with the hypothesis that the true mean equals zero (4).

Immunofluorescent Staining for Proliferation

To determine whether proliferation of cells in the melanocyte lineage occurred throughout the irradiation period, we co-stained for Ki-67 and ΔNp63 (Fig. 5H). As previously shown by immunohistochemistry, an increasing proportion of epidermal keratinocytes expressed Ki-67 throughout the 7 weeks of radiation treatment (40). In contrast, all cells in the melanocyte lineage (ΔNp63-negative cells) were negative for Ki-67. Thus, proliferation is not indicated in the melanocyte population over 7 weeks of radiation treatment and proliferation cannot explain the significant increase in the number of MITF-positive and Bcl-2 positive cells upon ionizing radiation exposure described above.

Immunofluorescent Staining of p53 and p21

We double-stained p53 and ΔNp63 to reveal any upregulation of p53 (Fig. 5I). The expression of p53 in epidermal keratinocytes was previously shown by immunohistochemistry to increase throughout radiation treatment (40), which was also obvious in the current double-staining. However, all ΔNp63-negative cells were also negative for p53. Thus, the melanocyte population did not express p53 upon ionizing radiation exposure.

Upregulation of p53 in keratinocytes is associated with the transcription of p21. The results of co-staining for p21 and ΔNp63 are shown in Fig. 5J. In agreement with the lack of p53 expression in melanocytes, no nuclear p21 staining was detected in ΔNp63-negative cells, though we did observe weak staining in the cytoplasm. However, upregulation of p21 was evident in keratinocytes as shown in previously published studies (4, 40).

FIG. 4.

The number of Bcl-2-positive vs. MITF-positive cells for all 33 individual patients in unexposed control samples and for each dose of fractionated radiation. The correlations are significant (P < 0.001).

Activation of c-KIT and DCT

Co-staining for c-KIT and ΔNp63 revealed that c-KIT-positive cells were found exclusively among the ΔNp63-negative cells. Approximately one half of the cells in unexposed skin were c-KIT-positive, and the number increased gradually at 1 and 6.5 weeks of radiotherapy (Fig. 5K). Double-staining for c-KIT and MITF showed that in unexposed skin, all MITF-positive cells were c-KIT-positive and all MITF-negative cells were also c-Kit-negative (data not shown).

Double-staining for DCT and ΔNp63 showed that approximately one half of the ΔNp63-negative cells in unexposed skin were DCT-positive. The number of DCTpositive cells increased gradually with 1 and 6.5 weeks of radiation treatment. After 6.5 weeks, almost all ΔNp63-negative cells were DCT-positive (data not shown). Furthermore, DCT double-stained with MITF confirmed an increase in the number of MITF-positive/DCT-positive cells over the course of radiation treatment. At the end of treatment, almost all cells were stained for MITF and DCT. In unexposed skin, MITF-negative cells were DCTnegative, but MITF-positive cells also expressed DCT to some extent (Fig. 5L). After exposure, the melanocytes expressed higher levels of DCT and increase in the number of dendrites.

FIG. 5.

Double-staining of epidermal skin biopsies illustrating the response of melanocytes to daily fractionated treatments of 1.1 Gy. Cells in the melanocyte lineage are exclusively negative for ΔNp63. Each double-staining was merged with DAPI. Panel A: Double-staining for ΔNp63 and MITF. Before radiotherapy, the left arrows indicate an MITF-negative melanocyte and right arrows indicate a MITF-positive melanocyte in the basal layer. During radiotherapy, the arrows indicate MITF-positive melanocytes. Panel B: Double-staining for ΔNp63 and Bcl-2. Before radiotherapy, the left arrows indicate a Bcl-2-negative melanocyte and right arrows indicate a Bcl-2-positive melanocyte in the basal layer. During radiotherapy, the arrows indicate a Bcl-2-positive melanocyte. Panel C: Double-staining for MITF and Bcl-2. Arrows indicate Bcl-2 cells co-expressing MITF and vice versa before and during radiotherapy. Panel D: Double-staining for MITF and SOX10. Before radiotherapy, the left arrows indicate a MITF-negative/SOX10-positive melanocyte in unexposed skin. During radiotherapy, the arrows indicate a MITF-positive/SOX10-positive melanocyte. Panel E: Double-staining for ΔNp63 and PAX3. The arrows indicate ΔNp63-negative/PAX3-positive melanocytes before and during radiotherapy. Panel F: Double-staining for MITF and PAX3. Before radiotherapy, the arrows indicate a MITF-negative/PAX3-positive melanocyte. During radiotherapy, the arrows indicate melanocytes that co-express MITF and PAX3. Panel G: Double-staining for PAX3 and SOX10. Before radiotherapy, the arrows indicate a PAX3-negative/SOX10-positive melanocyte. During radiotherapy, the left arrows indicate a PAX3-positive/SOX10-negative melanocyte and right arrows indicate a PAX3-negative/SOX10-positive melanocyte. Panel H: Double-staining for ΔNp63 and Ki-67. The arrows indicate Ki67-negative/ΔNp63-negative melanocytes before and during radiotherapy. Panel I: Double-staining for ΔNp63 and p53. The arrows indicate p53-negative/ΔNp63-negative melanocytes before and during radiotherapy. Panel J: Double-staining for p21 and ΔNp63. The arrows indicate nuclear p21-negative/ΔNp63-negative melanocytes before and during radiotherapy. Panel K: Double-staining for c-KIT and ΔNp63. The arrows indicate ΔNp63-negative/c-KIT-positive melanocytes before and during radiotherapy. Panel L: Double-staining for DCT and MITF. The arrows indicate DCT-positive/MITF-positive melanocytes before and during radiotherapy.

Quantification of Melanocytes by ΔNp63 and Eosin-PAS Staining

ΔNp63, an isoform of p63, is a key lineage-specific determinant of stratified epithelia, such as that of the epidermis (41–44). ΔNp63 is the predominant p63 isoform identified to date. Although total p63 is minimally expressed in normal melanocytes (45), melanocytes express small amounts of two of the six isoforms of p63, which have not been definitively determined. Importantly, none of the p63 isoforms are induced by ionizing radiation or UVR (36). We used the 4A4 anti-p63 monoclonal antibody in a manner similar to other studies and found intense staining of keratinocytes in the basal and suprabasal cell layers (Fig. 1A). The ΔNp63 staining was of variable intensity in keratinocytes (Figs. 1A, 5A, 5B, 5H–K) and comparable to previously reported findings (41). In light of the intense p63 staining, we cannot exclude the possibility that a small amount of p63 expression in melanocytes may serve as a confounding factor leading to a slight underestimation of the number of melanocytes (Table 1, Fig. 2A). By using decreasing concentrations of the 4A4 anti-p63 monoclonal antibody (from 1:100 up to 1:4,000), we found that 1:800 would have been the most optimal concentration, giving a 10% higher number of ΔNp63-negative cells than the 1:200 concentration used in this study (data not shown).

MITF-mediated activation of melanin-synthesizing enzymes requires the chromatin remodeling SWI/SNF complexes (46), which are necessary for MITF-based regulation of melanocyte differentiation. MITF recruits SWI/SNF complexes to the promoters of melanocyte-specific genes responsible for melanin synthesis, where the SWI/SNF enzymes remodel the chromatin to activate gene expression. Chromatin remodeling by the SWI/SNF complexes may induce the morphological changes in cells of the melanocyte lineage that were observed by eosin-PAS staining after lowdose irradiation (Fig. 1). Undifferentiated cells in unexposed skin are not visualized as easily by eosin-PAS staining as those in irradiated skin, which may lead to an underestimation of their number in control biopsies. This scenario is the most likely explanation for the significant increase in melanocytes after irradiation in the morphological evaluation (Table 1 and Fig. 2B).

Despite the confounding factors considered for the current ΔNp63 and eosin-PAS staining, the accuracy of the determined numbers of interfollicular melanocytes is satisfactory due to the strong correlation between the two estimations for individual patients in both unexposed and irradiated skin (Fig. 3).

Individual Variability in Melanocyte Density Assessed by Various Molecular Markers

Very little is found in the literature that addresses the density of cutaneous melanocytes. Barlow et al. determined the density of epidermal melanocytes by H& E staining of 6-µm sections; at 40× magnification the mean ± SD number of cells was 7.96 ± 6.7 cells/mm (35). No difference in melanocyte density was found between skin samples taken from regions of the head, neck, trunk and extremities. Furthermore, no difference in melanocyte density was found between skin types. Barlow et al. also reported a high degree of individual variability in melanocyte density, ranging from 0.7 to 33.4 cells/mm of epidermis [coefficient of variation (CV) 84%]. Tadokoro et al. (47) determined the melanocyte density in the basal layer to be 12.8 ± 1.2 melanocytes/mm by counting cells positive for MITF, MART-1, Pmel17 and tyrosinase. Shin et al. (17) reported 19.6 ± 5.7 cells/mm for the MITF-positive melanocyte density in normal skin. In these studies, no particular considerations were mentioned regarding the history of sun exposure for the investigated skin.

In the current study, the interfollicular epidermal melanocyte density was determined comprehensively. In unexposed skin, we counted 17.4 and 18.4 melanocytes/mm of epidermis for ΔNp63-negative cells and morphologically assessed melanocytes (eosin-PAS staining), respectively. The corresponding ranges in patients were 12–24 cells/mm for ΔNp63 staining and 12–27 cells/mm for eosin-PAS staining; the CVs were 22% and 20%, respectively.

The maximum number of melanocytes determined in the MITF and Bcl-2 staining experiments after irradiation were 20.9 and 21.9 cells/mm, respectively. The CV for MITF number was 27% and for the Bcl-2 number 23%. The individual variability in melanocyte density, though rather limited in our assessment, was confirmed by the strong correlation between Bcl-2-positive and MITF-positive melanocytes in unexposed skin and irradiated skin (Fig. 4). Collectively, individual variability in melanocyte density was threefold lower in our study than reported by Barlow et al. (35).

In the patient cohort presented here, the number of basal keratinocytes was determined to be 160 cells/mm (4). Based on all staining experiments, the number of melanocytes was 18–23 cells/mm, indicating that cells in the melanocyte lineage comprise close to 13% of the cells in the basal cell layer of the interfollicular epidermis. These data are associated with a Swedish male population usually presenting with skin types 2 and 3; skin types 1 and 4 are rare in this population (48, 49). Furthermore, the assessments were performed using skin biopsies taken from the hip region, which is not usually exposed to sun. No significant correlation between patient age and melanocyte density was found in the range of 49 to 74 years.

Quantification of MITF and Bcl-2

The number of MITF-positive cells gradually increased with daily dose per fraction of 0.05–1.09 Gy when assessed at 1 and 6.5 weeks of radiotherapy, with similar dose responses at the two time points (Table 1). The number of MITF-negative melanocytes decreased in a dose-dependent manner within the same dose range and exhibited hyperradiosensitivity to fractionated doses as low as 0.05 Gy (Table 1). The decrease in MITF-negative cells with dose precisely reflects the increase in MITF-positive melanocytes and indicates hypersensitivity to differentiation at doses below 0.3 Gy (Fig. 2C). Co-staining for ΔNp63 and MITF demonstrated the existence of MITF-negative cells, i.e., undifferentiated melanocytes, in the interfollicular melanocyte population and their disappearance after irradiation (Fig. 5A).

Similarly, the number of Bcl-2-positive melanocytes increased gradually with daily dose per fraction of 0.05–1.09 Gy, with a similar dose response at 1 and 6.5 weeks of radiotherapy (Table 1). In addition, the number of BCL-2-negative melanocytes decreased and hyperradiosensitivity was demonstrated at doses below 0.3 Gy (Fig. 2D). The connection between MITF and Bcl-2 is expected from in vitro studies showing that MITF promotes cell viability by regulating Bcl-2 (25). Here, we confirmed that this regulation persists in the clinical setting of radiotherapy, as illustrated by the strong correlation between Bcl-2 and MITF levels in each patient (Fig. 4). This observation was confirmed by the one-to-one correspondence in MITF and Bcl-2 co-staining (Fig. 5C).

Apoptosis and Proliferation

Melanocyte resistance to genotoxic stress has been attributed to upregulation of anti-apoptotic protein Bcl-2 by MITF (25). In addition, melanocytes have been confirmed to be resistant to UVR-induced apoptosis mediated by the activation of MITF in association with SWI/SNF complexes (46, 50). Importantly, the SWI/SNF complexes prevent DNA damage-induced apoptosis, an effect that is related, at least in part, to the ability of SWI/SNF complexes to repair DNA DSBs (51). Thus, the SWI/SNF complexes exert strong effects on the fate of melanocytes by driving differentiation and preventing apoptosis after genotoxic insult.

In a previously reported evaluation of apoptosis in the basal cell layer, only 0.2–1.5 apoptotic cells/mm was detected in the patient groups; the highest rate was observed after receiving 1.1 Gy dose fractions for 6.5 weeks (4). Therefore, we conclude that apoptosis in the epidermal melanocyte population is negligible. Notably, in a numerical comparison, cell death by apoptosis cannot explain the disappearance of MITF- and Bcl-2-negative cells upon exposure to ionizing radiation.

Ki-67 staining allowed us to exclude the possibility of significant melanocyte proliferation during the 7 weeks of treatment, supporting the consistent number of ΔNp63-negative cells observed during treatment (Fig. 2A). We can also state that the increase in MITF- and Bcl-2-positive cells upon ionizing radiation exposure is not caused by proliferation.

Differentiation Status of Skin Melanocytes

In this study we conclusively show that a subset of interfollicular melanocytes in unexposed skin is MITFnegative; these were also DCT-negative and c-KITnegative. In this subset, we identified PAX3-positive and SOX10-positive cells. To the best of our knowledge, this finding is unique. We believe that the main reason that we could reveal a subset of MITF-negative melanocytes is that all biopsies were taken from the hip region, which is not usually exposed to sun. Furthermore, we established doseresponse relationships showing how the number of MITFnegative cells decrease and the number of MITF-positive cells increase after exposure for each individual patient. This finding suggests a prompt need to further characterize the subset of MITF-negative cells among interfollicular melanocytes; in particular, if melanocyte stem cells do exist or if the undifferentiated cells are transit amplifying cells migrated from the hair bulge.

In the hair follicular bulge area of human skin the melanocyte stem cells are DCT⁺c-KIT^– and the melanoblasts are DCT⁺c-KIT⁺, but DCT^–c-KIT⁺ cells have also been identified. Subsets of melanocyte precursor cells in the bulge area are PAX3-positive and SOX10-positive. MITF expression occurs only transiently (10, 12). In the interfollicular epidermis, subsets of cells express DCT, c-KIT, PAX3, SOX10 and MITF. Co-expression of the various markers is not well known. However, a variable differentiation status among interfollicular melanocytes were identified by Medic and Ziman (15), showing that approximately 20% of the melanocytes were less differentiated.

Radioresistance Induced by Differentiation

In the current study, we established that melanocytes are radioresistant regarding cell death and remain constant in number to fractionated doses below 1.1 Gy over the 7 weeks of treatment. The upregulation of MITF induced by UVR requires activation of PAX3, SOX10 and c-AMP. UVRA induced c-KIT activity also promotes MITF expression. We established that increased expression of both PAX3 and SOX10 occurs in parallel with upregulation of MITF after irradiation and also observed an increase in c-KIT. Further evidence of differentiation after irradiation was established by staining for DCT, which revealed increased numbers of DCT-positive cells, enhanced staining intensity and higher density of enlarged dendrites after irradiation (Fig. 5L). Taken together, the first steps of radiation-induced melanin synthesis were confirmed.

The double-staining experiments with SOX10 and MITF, PAX3 and MITF, SOX10 and PAX3, and MITF and DCT that were used in our current study suggest similar regulation of MITF transcription after irradiation and what has already been established for UVR exposure, with the initiation of differentiation as the major response mechanism (Fig. 5D, F and G).

In support of our findings, differentiation as a response to ionizing radiation was confirmed for melanocyte stem cells in the hair follicle bulge of adult mice (29). After 5 Gy irradiation, melanocyte stem cells lose their immaturity and renewal to become melanin-producing dendritic melanocytes.

Keratinocyte-Melanocyte Interactions

Upon exposure to ionizing radiation, nuclear expression of p53 and p21 was very weak and considered negative in melanocytes compared to surrounding keratinocytes (Fig. 5I and J). This phenomenon was reported previously for human skin after exposure to UVR (52, 53), but the molecular pathways that suppress the p53-p21 pathway in melanocytes is poorly understood. However, PAX3 was recently shown to inactivate the function of p53 by stimulating its ubiquitination and degradation (54). Furthermore, ATR function is suppressed in melanocytes after UVR exposure and chemotherapy (cisplatin), suggesting a lack of activation of p53 and, subsequently, p21 (28). This particular response in melanocytes is also expected after radiation-induced DNA damage. Interestingly, ATR inhibition was confirmed to protect noncycling human cells exposed to UVR from undergoing apoptosis (55). Specific regulation of ATM kinase in melanocytes is still not known.

In a previously published study, although p21 mRNA and protein levels increased after DNA damage to melanocytes, p53 binding to p21 promoter DNA was undetectable (36). The lack of p53 and p21 expression in melanocytes in the current study highlights the critical role of ionizing radiation-induced p53 expression in keratinocytes and its associated paracrine signaling to melanocytes. The dosedependent ATM-driven paracrine signaling from keratinocytes is reflected in the hypersensitive dose-dependent increase in MITF expression by melanocytes in parallel with the hypersensitivity found for all end points studied in keratinocytes (4).

We suggest that the hypersensitivity response toward melanocyte differentiation reflects incomplete phosphorylation of ATM in keratinocytes over the low-dose range (56) and corresponding incomplete dose-dependent paracrine signaling from keratinocytes. It has been previously established that skin keratinocytes exhibit hypersensitivity below 0.3 Gy for DNA DSBs, growth arrest, mitosis, apoptosis and overall cell loss (2–4).

In conclusion, constant levels of ΔNp63-negative cells suggest that the number of cells in the melanocyte lineage is preserved throughout the fractionated radiation therapy over 6.5 weeks at doses up to 1.1 Gy. A subpopulation of undifferentiated MITF- and Bcl-2-negative cells exists in unexposed interfollicular epidermis. After exposure to low doses of ionizing radiation, this subset of cells upregulates MITF and Bcl-2 in association with increased expression of PAX3, SOX10, c-KIT and DCT, indicating that differentiation and radioresistance for cell death is induced. The results suggest that the initial steps of melanin synthesis are common after exposure to ionizing radiation and UVR.