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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Near infrared (NIR) and X-rays are radiations from different sides of the wavelength spectrum but both are used during medical treatments, as they have severe impacts on cellular processes, including metabolism, gene expression, proliferation and survival. However, both radiations differ strictly in their consequences for exposed patients: NIR effects are generally supposed to be positive, mostly ascribed to a stimulation of metabolism, whereas X-ray leads to genetic instability, an increase of reactive oxygen species (ROS) and DNA damages and finally to cellular death by apoptosis in tumor cells. Since genomic stability after X-irradiation depends on the mitochondrial metabolism, which is well known to be regulated by NIR, we analyzed the impact of NIR on cellular responses of fibroblasts, retinal progenitor cells and keratinocytes to X-radiation. Our data show that previous exposure to naturally occurring doses of nonthermal NIR combined with clinically relevant X-ray doses leads to (1) increased genomic instability, indicated by elevated ratios of mitotic catastrophes, (2) increased ROS, (3) higher amounts of X-irradiated cells entering S-phase and (4) impaired DNA double-strand break repair. Taken together, our data show tremendous effects of NIR on cellular responses to X-rays, probably affecting the results of radiotherapy after NIR exposure during cancer treatment.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Forty percent of the energy from solar radiation reaching the earth’s surface is contributed by near-infrared (NIR) radiation, to which man and all organisms are permanently exposed during everyday life. Since hemoglobin, water and lipids as the main absorbers of light have their lowest absorption coefficient in the NIR range, NIR is able to penetrate deep into living tissues, thereby stimulating the basic energy processes in the mitochondria of cells within the exposed area. This is due to the activation of cytochrome c oxidase (COX) as the main absorber of NIR, ending up in an increase in the respiratory metabolism of certain cells (1,2). Furthermore, COX might act as a photosignal transducer within the retrograde signaling pathway between mitochondria and the nucleus, thereby regulating gene transcription (2,3). In fact, exposure of fibroblasts to red light has been shown to regulate the transcription of at least 111 genes of 10 functional categories, whereby seven of these categories were directly or indirectly involved in cell proliferation. The remaining function categories were genes related to transcription factors, inflammation and cytokines as well as some genes not characterized (4). Independent from all these facts, NIR exposure has been deemed a nonsignificant risk by the U.S. Food and Drug Association (FDA), thus FDA approval for the use of LEDs (light emitting diodes) in humans for light therapy has been obtained (5,6). Due to this, NIR has become popular for medical applications, gaining more and more relevance for therapeutic purposes. It has been shown to influence wound healing (6) and angiogenesis in skin, bone and skeletal muscle (7–10). Finally, NIR enhanced the cell survival in UV-exposed human fibroblasts, due to the upregulation of antiapoptotic genes 24 h after the exposure. On the diagnostic side, NIR has become popular for noninvasive in vivo fluorescence imaging by NIR optical spectroscopy (11).

Contrary to NIR, ionizing X-radiation is used at low doses for “diagnostic radiography” (1–10 mGy) and at high doses (fractionated irradiation leading to local doses of up to 80 Gy) during cancer radiotherapy. Due to its ionizing character, it produces not only reactive oxygen species (ROS), but also DNA single- and double-strand breaks (DSBs) within exposed cells. Therefore, X-radiation elicit a variety of cellular damage responses, including cell cycle checkpoints that regulate entry or progression into mitosis, terminal growth arrest by senescence or cellular death by apoptosis and/or necrosis. As another consequence of X-irradiation, cells might undergo so-called mitotic catastrophes (MCs) due to aberrant mitoses. Thereby, disturbances in spindle and centrosome formation end up in the emergence of polynucleated and polyploid cells (12,13), which have recently been shown to represent an important endpoint to analyze after irradiation: cells that underwent MC are capable of producing viable and proliferative daughter cells with a disarranged genomic composition by meiosis (14–17). Such depolyploidizations can also be observed in solid tumor and circulating tumor cells following radiation-induced MC (18), suggesting that such a mechanism could underlie tumor progression and resistance to its treatment in vivo (15).

Due to these massive impacts of X-radiation on cells and tissues, the success of X-ray therapy depends decisively on a finely tuned design of optimally balancing the applied X-ray doses in combination with their locally restricted application. Any noncontrollable side effects might either lead to an ineffective destruction of tumor cells, or to deleterious effects on healthy neighboring tissues. Therefore, any parameters that could influence the effectiveness of an X-ray therapeutic design regarding genetic stability and cellular survival in a positive, or a negative manner are of highest clinical relevance. Such parameters might not only be the actual expression of apoptosis-related genes (19), but also the metabolic state—as a function of the mitochondrial status—of cells during X-irradiation (20,21).

Therefore, we analyzed the interactions between NIR as an agent that regulates metabolism and the expression of apoptotic and antiapoptotic genes, with ionizing X-radiation. NIR doses used in our experiments correspond to doses that can be received between 15 and 30 min exposure to solar radiation within midday hours, depending on solar intensity and geographical position (22-year average in June: 14 min Rome, Italy; 18 min New York, USA; 24 min Frankfurt, Germany; 30 min Sydney, Australia; data from the Atmospheric Science Data Center of NASA; see Material and Methods). They are also within the range of doses used during low level light therapy (LLLT) for deeper-seated disorders (100–500 kJ m−2) (22). X-radiation doses used within our experiments are comparable to doses which are applied as single fraction doses (2–4 Gy) or which are accumulated after several fractionated irradiations (10 Gy) during radiotherapy (23).

The data presented here provide the first in vitro experimental evidence that NIR is able to affect the response to X-radiation within mammalian cells of different origins, including human keratinocytes. Thus, our results support and extend data from the 1950s (24–28), which showed increased lethal or visible mutations in plants and Drosophila after combined NIR and X-ray treatment, and—most importantly—show their high relevance for human health.

Together, previous NIR treatment followed by different doses of X-rays resulted in (1) a higher number of MCs, (2) a higher level of ROS, (3) an increase of cells entering S-phase, as detected by BrdU uptake and (4) impaired repair of DNA damage shown by γH2AX studies in the mouse embryonic fibroblast cell line NIH-3T3, in human keratinocytes (HaCaT) and the rat retinal precursor cell line R28 (29). Thus, our data provide serious indications of an altered reaction to X-irradiation in cells that have been pre-exposed to naturally occurring, or, medically applied NIR during radiotherapy.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Irradiation.  NIR irradiation was basically performed as described by Menezes et al. (30). Samples in irradiation medium (DMEM, free of carbonate, supplemented with 10 mm HEPES) were placed in a water-cooled plate, maintaining the temperature between 20 and 25°C. A 4 mm thick frosted glass and a 20 mm ice-cooled water filter were interposed between IR lamp (Philips IR250RH) and sample providing homogeneous irradiation and removing wavelengths higher than 1400 nm. The irradiance at the cells plane was 20 mW m−2 measured with an IL1700 Thermopile Detector and Research Photometer (International Light). After 30 min a fluency of 360 kJ m−2 was reached. Irradiation with X-rays (90 kV, 33.7 mA, 5.23 Gy min−1) was performed in culture media; thereby we considered that cells X-irradiated on glass slides receive a higher dose as determined by physical and chemical dosimetry (31). After irradiation, the media was supplemented (when indicated) with 25 μm BrdU.

Cell culture.  R28 and HaCaT cells were cultured in DMEM, supplemented with 10% FCS, 2 mm l-glutamine, penicillin and streptomycin. Culture media for NIH/3T3 mouse fibroblasts was further supplemented with 1% NEAA. All cells were used for less than 6 months after resuscitation to gain the described results.

The original authentication of the R28 stock was done by Gail M. Seigel as follows: R28 cells are a retinal precursor cell line developed from postnatal day 6 rat retina immortalized with the 12S gene of Adenovirus E1A (29) and developed into a clonal cell line by limiting dilution. They have been used in over 30 publications and have been characterized by microarray analysis (32) and also by functional responses to neurotransmitters (33). A number of papers have examined these cells using rat-specific probes, which indicates that they are of rat origin. R28 retinal precursor cells were kindly provided by Gail M. Seigel in 2005. Resuscitation of this stock was done in October 2009 and has been regularly checked before each experiment according to morphological criteria. Chromosome spreads have been additionally done for identification of rat karyotype.

NIH/3T3 cells, kindly provided by Steffen C. Naumann, were originally obtained from DSMZ (ACC 59) in March 2009. Species characterization at DSMZ was confirmed as mouse with IEF of AST, MDH, PEP B and by species PCR. Resuscitation was done in March 2010. Further the cells have been regularly checked before each experiment according to morphological criteria, RT-PCR was performed with mouse specific primers (see also Figure S1) and chromosome spreads have been done for identification of mouse karyotype.

Human keratinocytes HaCaT were kindly provided by Beate Volkmer (Elbekliniken Buxtehude) in March 2009. The original stock was established and characterized by Petra Boukamp (34). mFISH analyzes with human specific probes had been performed and published (35), verifying the human origin. Resuscitation was done in August 2009 and March 2010. Further the cells have been regularly checked before each experiment according to morphological criteria.

Immunohistochemistry.  Cells were fixed for 10 min in 4% paraformaldehyde, followed by washing in PBS buffer. Immunohistochemistry staining was usually performed by incubation with primary antibody (BrdU: 1:500, G3G4, DSHB; 8-oxo-dG: 1:200, 15A3; Santa Cruz Biotechnology, Inc., Germany; γH2AX: 1:500 antiphospho-Histone H2A.X [Ser139] antibody; Millipore) in blocking solution (5% BSA, 0.1% Trition X 100 in PBS), following by CY3-conjugated secondary antibodies (1:200; Dianova, Germany) and counterstaining of DNA with DAPI. For BrdU detection, samples were incubated in 2n HCl for 5 min, followed by neutralization in PBS prior to antibody staining.

Image analysis.  The image analysis of BrdU or 8-oxo-dG (8-oxo-7,8-dihydro-2′-deoxyguanosine) staining was carried out using a Zeiss Observer A1 microscope for epifluorescence, equipped with a CCD camera (Zeiss, Germany). Images were automatically taken and mean fluorescence intensity within DAPI-contour was analyzed by MetaCyte software (Metasystems, Germany).

Detection of MCs.  DAPI-stained nuclei were counted using a Zeiss Observer microscope for epifluorescence. Distinction of cells with micronuclei and MCs was achieved by using the criteria for micronuclei as established by Fenech (36). Cells with polynuclei, not fitting to this criteria and with typical degenerated nuclei as described by Ianzini et al. (37), were rated as MCs.

Detection of mitotic indices.  DAPI-stained mitoses (prometa-, meta-, ana- and early telophases) were counted in 1000 cells per dose and replicate using a Zeiss Observer microscope for epifluorescence.

Detection of apoptotic cells.  Staining for TUNEL (TdT-mediated dUTP nick end labeling) positive cells was performed according to manufacturer’s manual (Promega, Germany) and counterstained with DAPI. Early vs late apoptotic events were determined by AnnexinV FACS analyses (38). Cells were incubated with AnnexinV-FITC reagent (PromoKine, Germany) in Annexin-binding buffer and counterstained with propidium iodide (100 ng μL−1) according to manufacturer’s manual.

Clonogenic cell survival assays.  Defined single cell suspensions of irradiated cells were prepared and at low density seeded into Petri dishes. After an incubation period of up to 10 days cultures were fixed and Giemsa stained. Colonies with more than 15 cells were counted and survival was normalized to nonirradiated controls of sham and NIR-pretreated cultures. Each dose was analyzed in several independent experiments in triplicate.

Cell cycle FACS analyses.  Cells were harvested and single cell suspensions were fixed in 70% ice cold ethanol. After washing in PBS cells were stained 30 min in propidium iodide (30 μg mL−1) supplemented with RNase (0.5 μg μL−1) and FACS analyses for DNA content were performed.

FACS analyses of mitochondrial mass.  In order to analyze the impact of NIR on the mitochondrial status (39–41), cells were incubated in 100 nmMitoTracker Green® (Invitrogen, Germany) in culture media for 10 min at 37°C. After washing in PBS, FACS analyses were performed.

FACS analyses of ROS.  Prior to X-ray irradiation, cells were incubated in 25 μm DCFDA (Sigma-Aldrich, Germany) in PBS for 30 min at 37°C. After washing in PBS cells were irradiated and FACS analyses were performed at certain time points after irradiation.

Western blot analysis.  Cells were washed with PBS, pelleted and lysed in Laemmli buffer (0.125 m Tris-HCl, pH 6.8, 5% SDS) containing Complete™ (Roche Molecular Biochemicals, Germany) and 1 mm Na3VO4. Aliquots of 80 μg protein plus 5%β-mercaptoethanol were size-fractionated on SDS-PAGE and electroblotted onto nitrocellulose membranes (Millipore). After blocking of nonspecific binding sites with 5% nonfat dried milk, membranes were incubated with primary antibodies (antiphospho-Histone H2A.X [Ser139] antibody, 1:2000, Millipore; anti-GAPDH antibody, 1:1000, Santa Cruz Biotechnology, Inc.). Antibody binding was detected with secondary HRP-antibodies (1:10 000, Santa Cruz Biotechnology, Inc.) and LumiLight (Roche Molecular Biochemicals). Bands were quantitated with ImageJ.

RNA isolation and RT-PCR analysis.  RNA of harvested cells was isolated using MasterPureTM Complete DNA and RNA Purification Kit (Epicentre). cDNA syntheses were performed with Revert Aid First Strand cDNA-Kit (Fermentas, Germany) according to manufacturer’s manual using oligo(dT)18 primer. PCR amplifications were performed with TopTaq Polymerase Kit (Qiagen, Germany) using following primers for each genes of interest: hsp27 (f: 5′-GCCGCACCAGCCTTCAGC; r: 5′-CACGCCTTCCTTGGTCTTCACT) and hsp70 (f: 5′-GCTGCTTGGGCACCGATTACTGT, r: 5′-CTCCAGGGAACTGGGCAGCTAGA).

Calculation of global NIR exposure.  Global NIR exposure was calculated using data from ASTM G173-03 Reference Spectra Derived from SMARTS v. 2.9.2, provided by the National Renewable Energy Laboratory (NREL) of the U.S. Department of Energy (http://www.nrel.gov/rredc/), showing a NIR ratio (corresponding to wavelengths of 660–1400 nm, as supplied by our radiation source) of 49% within solar radiation reaching the earth surface. This value was taken for the further calculation of exposure time, needed for the accumulation of 360 kJ m−2 NIR within different countries during midday hours in June, based on average data for the last 22 years, which were taken from the Atmospheric Science Data Center of NASA (42).

Statistics.  Statistics were performed using GraphPad Prism statistical software (version 3.02). If not indicated otherwise 1000 cells per independent experiment (n) for each dose were scored for statistical analyses. Unpaired two-tailed student’s t-test was used for quantification of BrdU, mitotic indices and γH2AX analysis (50 cells for all X-irradiated samples, 100 cells for others). One-way ANOVA with Tukey-test was used for MC-long term studies, for TUNEL analysis (500 cells), 8-oxo-dG (250 cells) and FACS analysis (30 000 cells). Chi-square test was used for P-value calculation of MC studies. For all analyses a CI = 95% with *P < 0.05, **P < 0.01 and ***P < 0.001 was defined.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

NIR pretreatment strongly increase X-ray induced MCs

MCs (shown in Fig. 1a–d) are an indicator for mitotic failure and genetic instability in specially treated cells (Fig. 1a,b: R28 cells; 1c,d: HaCaT cells). To analyze the impact of NIR treatment alone or in combination with X-rays, ratios of MCs were counted 24 h after exposure to ionizing radiation.

image

Figure 1.  DAPI-stained polynucleated cells, not fitting the criteria established by Fenech (36), characterize mitotic catastrophes (MCs), (a–c in G-phase; d in metaphase; cell outlines indicated by dashed lines) and were used here as an endpoint to indicate genomic instability. Note that MCs result from aberrant mitoses leading to polynucleated or polyploidy cells (see text and [37,48,68,73,74]). (a, b) R28 cells; (c, d) HaCaT cells; bar = 20 μm.

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Figure 2 presents the ratios of MCs in three cell lines after increasing doses of X-rays, either without or with NIR pretreatment of 360 kJ m−2. MCs were almost undetectable in sham-exposed as well as in NIR-treated cells in all cell types. In contrast, in fibroblasts and R28 progenitors MCs increased with ascending doses of X-rays (Fig. 2a,b; black bars). In keratinocytes, the most effective dose was at 4 Gy (Fig. 2c).

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Figure 2.  Near infrared (NIR) pretreatment increases the generation of mitotic catastrophes (MCs) after irradiation with X-ray. Ratios of MCs increased with ascending X-ray doses (black) in 3T3 fibroblasts (a), R28 retinal precursor cells (b) and HaCaT keratinocytes (c) 24 h after irradiation. Note that NIR treatment alone (left gray bars) did not alter MC numbers, but when followed by X-ray (middle and right gray bars), significantly more MCs appeared after 4 Gy when compared with X-ray alone in fibroblasts (a), after 4 Gy in R28 cells (b) and after 10 Gy in keratinocytes (c). Further ratios of MCs were determined 24 (black), 48 (gray) and 72 h (white) after 4 Gy of X-radiation in R28 cells (d) and 10 Gy in keratinocytes (e). Generation of MCs did not stop after 24 h, but was further increasing after 48 h in both cell lines and even after 72 h in R28 cells. Note that NIR treatment alone did not alter ratios of MCs. Data are represented as means (a, c: 3T3/HaCaT: n = 4; b: R28: 2 Gy n = 4, others n = 7; d, e: n = 3) ± SE (*P < 0.05, **P < 0.01, ***P < 0.001).

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Most remarkably, NIR treatment before X-radiation resulted in an even higher increase of MC, when compared to the exclusive X-irradiation. 3T3 fibroblasts showed a slight but significant increase after 4 Gy and an almost doubled amount of MCs after 10 Gy (Fig. 2a). For NIR-exposed R28 progenitor cells, a slight increase after 2 Gy and more than a doubling of MCs after 4 Gy was documented (Fig. 2b). Similar differences were obtained for HaCaT after 4 and 10 Gy (Fig. 2c).

To clarify whether MCs are formed only during the first 24 h after X-ray irradiation, the ratios of MCs were also determined after 48 and 72 h in both R28 and HaCaT cells (Fig. 2d,e). Thereby, only X-ray doses were applied which effectively induce MCs in the corresponding cell line. In absence of X-rays, levels of MCs remained very low in both cell types of sham-exposed and NIR-treated cells over the entire period (Fig. 2d,e). MCs were continuously amassed after X-rays alone (4 Gy in d; 10 Gy in e). Most remarkably, in both cell lines and at all time points, a NIR pretreatment led to an even further increase in the rate of MCs (Fig. 2d,e).

Since aberrant mitoses are assumed to cause MCs (see Introduction), the total number of cells capable to still undergo mitosis after a first NIR irradiation will be crucial for the degree of MC formation after a second treatment with X-rays. Therefore, we determined cell cycle progression by FACS analysis in 3T3 fibroblasts and the mitotic indices of 3T3 fibroblasts and HaCaT cells 24 h after NIR treatment. In both analyses, no significant deviations from controls (-NIR) could be detected. This surprising finding indicates that the same ratio of dividing cells would become irradiated by a subsequent X-ray treatment. In other words, the combined effect of NIR and X-ray irradiations on MC formation (see above) cannot be due to different ratios of cells undergoing mitosis at the time when cells become exposed to X-rays (further, see Discussion).

NIR pretreatment does not affect apoptosis but changes the ratio of clonogenic survival

Since X-irradiation is able to cause severe DNA damages, which finally can lead to cell death, we investigated influences of single and combined irradiation regimes on the survival of cells (Fig. 3). First, the ratios of TUNEL-labeled cells were determined (Fig. 3a). In both retinal progenitors and 3T3 fibroblasts, less than 1% of TUNEL+ cells could be detected after either treatment with no significant differences between them. We then performed FACS analyses after AnnexinV and propidium iodide labeling in fibroblasts, which allowed us to distinguish early apoptotic from late apoptotic/necrotic events, respectively (Fig. 3b). Minor increases of both early and late events could be detected after X-ray irradiation, whereby a pretreatment with NIR did not make any difference. Both events were increased in a positive control using Actinomycin D treatment (Fig. 3b). This shows that a first irradiation with NIR does not affect the rate of apoptosis as induced by X-rays alone.

image

Figure 3.  Near infrared (NIR) pretreatment does not affect apoptotic events following X-ray irradiation. (a) FACS analyses of apoptotic events in fibroblasts by AnnexinV to indicate early apoptotic stages, and double labeling with propidium iodide for advanced apoptotic stages showed no effect for NIR treatment. Exclusive and NIR-combined X-ray treatment similarly decreased numbers of viable cells (black), with more early (gray) than late (white) apoptotic/necrotic events. (b) Differences of ratios of TUNEL (TdT-mediated dUTP nick end labeling) positive cells were insignificant for all treatments, both in fibroblasts (left) and R28 cells (right, light gray: 24 h after irradiation with 4 Gy of X-ray; white: with pretreatment with 360 kJ m−2 NIR; dark gray: NIR treatment alone). Data are represented as means (n = 3) ± SE (*P < 0.05, **P < 0.01, ***P < 0.001).

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To analyze the impact of a combined treatment on clonogenic survival, we additionally carried out clonogenic cell survival assays with 3T3 fibroblasts and R28 retinal progenitor cells. Comparing the numbers of colonies formed by NIR- and sham-exposed cells after 4 and 10 Gy, we observed no changes within 3T3 fibroblasts, but a significant increase in clonogenic survival after 4 Gy for R28 progenitor cells: exclusive X-irradiation reduced the numbers of colonies formed to 41.8 ± 1.5% (SE) when compared to controls, whereas NIR pretreatment elevated the ratio up to 58 ± 1.3% (SE) (P < 0.001). However, no differences but still an overall survival of ca 31.5% for R28, and 11.5% for 3T3 fibroblasts after 10 Gy of X-radiation could be observed.

An exclusive NIR treatment alters the mitochondrial mass within the exposed cells

NIR is well known to interact with mitochondria (1–3). Genetic stability, which is obviously affected in the case of our combined radiation exposure, depends—amongst others—on mitochondrial metabolism of cells (20,21). Therefore, we analyzed the impact of NIR doses that were used in this study on the amount of mitochondria present within the cells 24 h after the NIR exposure. Cells were stained with MitoTracker Green® as a fluorescent dye to monitor the mitochondrial status by FACS analysis (39–41). As indicated in Fig. 4, 3T3 fibroblasts (Fig. 4a) as well as R28 retinal progenitor cells (Fig. 4b) showed a significant increase in their mitochondrial masses, when compared to unexposed control cells. At the same time, no changes within the mitochondrial mass could be observed for HaCaT cells.

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Figure 4.  Pretreatment with near infrared (NIR) enhances mitochondrial mass in 3T3 fibroblasts and R28 progenitors, but not in HaCaT keratinocytes. FACS analyses of mitochondrial mass via MitoTracker Green fluorescence showed a significant increase 24 h after NIR treatment in the high proliferative fibroblasts (a) and in R28 cells (b). No increase could be detected in the low proliferative keratinocytes (c). Data are represented as means (n = 3) ± SE (*P < 0.05, ***P < 0.001).

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NIR pretreatment increases X-radiation-induced generation of ROS

Our studies show that NIR pretreatment leads to an increase of genomic instability after X-irradiation, while—at the same time—apoptosis is not affected. Since our NIR treatment alters the number of mitochondria present during X-irradiation (see Fig. 1), we measured the level of intracellular ROS levels that emerged after X-irradiation as an indicator for an altered-cellular signaling and also DNA synthesis (43) by DCFDA staining and subsequent FACS analysis in 3T3 fibroblasts (Fig. 5a). Comparing the levels of intracellular ROS between sham and NIR-exposed samples 24 h after NIR treatment, only a slight but not significant increase was observed in NIR-treated cells (P = 0.105). When sham-exposed cells were treated with 10 Gy of X-rays, we observed a 10-fold increase within the ROS levels 30 min after the irradiation treatment, with a slow decrease down to a four-fold increase after 3 h. Noticeably, combined NIR and X-ray treatment led to even higher ROS levels up to 2 h after X-irradiation (P < 0.01).

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Figure 5.  Pretreatment with near infrared (NIR) showed a more than additive effect on the level of endogenous reactive oxygen species (ROS) after X-ray irradiation. (a) ROS levels, analyzed by DCFDA fluorescence, in NIR-treated 3T3 fibroblasts (dark gray) showed a slight, but not significant higher ROS level (delta NIR; diagonal stripes) than nontreated controls (black). Irradiation of nontreated control cells with 10 Gy of X-ray resulted in a significant increase of ROS levels (light gray), whereas NIR-pretreated cells revealed a much higher ROS level, representing a much higher than a mere additive effect of NIR and X-ray (dotted box). This effect remains up to 3 h after X-irradiation. (b) In R28 progenitors 8-oxo-dG signals in nuclei were increased after NIR treatment (gray, left panel) when compared to controls (black, left panel). X-irradiation (black, right panel) leads to a further increase even exceeded by a combined treatment with NIR and X-ray (gray, right panel). Data are represented as means of normalized arbitrary fluorescence units [a.u.] (n = 3) ± SE (**P < 0.01, ***P < 0.001).

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To evaluate direct interactions of generated ROS with the DNA, 4 h after exposure of R28 cells to 4 Gy of X-rays, the formation of 8-oxo-dG (8-oxo-7,8-dihydro-2′-deoxyguanosine) DNA adducts was analyzed via immunohistochemistry. A significantly higher level of 8-oxo-dG could be found in NIR-treated samples compared to sham control (Fig. 5b). Their level was exceeded by X-irradiated samples, which again was beaten by a combined NIR and X-ray treatment.

NIR pretreatment counteracts the decrease of cell proliferation by X-rays

As described above, NIR pretreatment significantly influenced the levels of ROS that emerged after X-irradiation in fibroblasts. Since ROS are well known to influence the level of proliferation within cell populations, we determined the proliferation rates in the differently treated samples by BrdU-uptake studies during the next 24 h after X-irradiation. Analyzing the effects of an exclusive NIR treatment during this time period, we observed a dose-dependent decrease of BrdU incorporation in R28 progenitor cells: Increasing doses of 180, 360 and 720 kJ m−2 lead to a successive reduction of the measured BrdU signal within the nucleus of −12%, −20% and −42%, when compared to the untreated controls (see Figure S2). For further experiments, a dose of 360 kJ m−2 was used, revealing an even stronger downregulation of BrdU incorporation (−27%) in 3T3 fibroblasts after exclusive NIR treatment (see Fig. 6a). X-irradiation alone also decreased the rate of BrdU uptake in a dose-dependent manner: at 10 Gy, for fibroblasts BrdU uptake was lowered almost halved, and for R28 the BrdU signal was lowered approximately by one-third. Remarkably, the combination of two proliferation-decreasing treatments (NIR in a dose of 360 kJ m−2 and X-radiation) did not end up in an even stronger decline of proliferation. In contrast, NIR counteracted the effects of X-radiation in fibroblast after 10 Gy (Fig. 6a). Comparable results were obtained for R28 retinal progenitors (Fig. 6b).

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Figure 6.  Near infrared (NIR) pretreatment (gray) counteracts decline of proliferation caused by X-ray irradiation, both in fibroblasts (a, 10 Gy) and R28 cells (b, 4 Gy and higher). Cell proliferation was detected by BrdU uptake 24 h after irradiation with X-rays. Data are represented as means (n = 3) ± SE (**P < 0.01, ***P < 0.001).

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NIR pretreatment delays DNA repair

Ionizing radiation is known to cause serious damage to cells including DNA DSBs capable to activate cell cycle checkpoints. A powerful marker for quantification and analysis of the repair capacity of DNA DSBs is the phosphorylated H2AX histone (γH2AX). Therefore, γH2AX foci were counted in G1-phase 3T3 fibroblasts, either with or without NIR pretreatment, 4 h after their exposure to 4 Gy of X-rays. A small number of spontaneous foci were detected in controls as well as in NIR-exposed cells (Fig. 7b, 0 Gy). Irradiation with X-rays effectively induced γH2AX foci in G1-cells, but noticeably, NIR-pretreated cells showed significantly lower foci numbers (Fig. 7b, 4 Gy). To analyze whether this increase after NIR treatment was due to a higher foci induction or to a delayed DSBs repair, foci in G1-cells were counted 15 min after exposure to 1 Gy (Fig. 7a). Interestingly, no significant alterations between X-irradiated and double-treated cells could be detected, supportive of a delay in the repair processes.

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Figure 7.  Near infrared (NIR) pretreatment does not influence ratios of X-ray-induced DNA double-strand breaks (DSBs), but more DSBs persist after 4 h of repair. While, NIR pretreatment did not change DSBs after 1 Gy X-rays (a), significantly more foci still persisted 4 h after 4 Gy (b). Note that NIR treatment alone did not change γH2AX-labeled DNA DSBs in G1-phase nuclei. (c) Western blot analyses of samples irradiated with 10 Gy showed more γH2AX-signals after NIR pretreatment, as compared with X-irradiation alone. Again NIR treatment alone had no effect on the γH2AX-signal. GAPDH was taken as control protein. (d) Quantification of Western blot bands from three independent experiments showed that the increase in γH2AX-signal after NIR pretreatment is significant. Data are represented as means (n = 3; see Materials and Methods) ± SE (*P < 0.05, ***P < 0.001).

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Therefore, DSBs repair was further analyzed in 3T3 fibroblasts 4 h after their exposure to a 10 Gy X-ray dose and determination of their content of γH2AX protein by Western blot (Fig. 7c). Only background levels of γH2AX protein were detectable in control and NIR-treated samples, while in X-ray treated samples the NIR-pretreated sample presented a significantly increased level of this protein; this is further documented by quantification of their respective bands (Fig. 7d).

NIR treatment induced no stress response via heat shock proteins

To exclude any NIR treatment-related stress response via heat shock protein expression, we performed RT-PCR for hsp70 and hsp27 at 24 and 48 h after treatment. We could show that none of the tested heat shock proteins was expressed at 24 h after NIR treatment when X-radiation was performed. Even after 48 h no enhanced expression level could be detected (Figure S1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

In this study we analyzed the effects of a combined treatment with nonionizing NIR and ionizing X-radiation on intracellular ROS levels, proliferation, cellular survival and genomic integrity in the case of DNA DSBs repair and emergence of MCs in three cell lines from different origins. Surprisingly, with the exception of apoptosis, NIR showed a strong effect on all these endpoints when compared to an exclusive X-irradiation. Thus, our findings represent the first study about an impact of nonthermal NIR on the response of mammalian cells to ionizing X-radiation.

These results are of high significance, since the NIR radiation doses used for our experiments can be accumulated within 15–30 min of solar exposure (depending on solar intensity and geographical position; see Introduction) or during LLLT of deeper-seated disorders (100–500 kJ m−2) (22). Used X-radiation doses can be accumulated by single or fractionated radiation during radiotherapy (23).

Even more, all cell lines analyzed originate from tissues that can be reached by NIR (44,45) and which, in turn, might represent targets in radiation therapy, e.g. epidermal and dermal cells in the case of skin cancer and retinal progenitors during irradiation of retinoblastoma treatment (46,47). Thus, all of our analyzed cells might be exposed during everyday life to the two discussed radiation qualities, rendering our study as medically highly relevant.

Higher numbers of MCs are caused by impaired DNA DSBs repair and premature entry into S-phase

Our studies show that combined irradiation with NIR and X-ray leads to an increase in genomic instability, indicated by higher numbers of viable MCs that occur after the treatment. To rule out that this effect is due to hyperthermia, which is able to enhance the development of MCs after irradiation (48), we have used an experimental setup which prevents heating of the cells (see Materials & Methods). Furthermore, the induction of thermal stress was excluded by the analysis of the expression of heat shock proteins.

Another reason for the increase of MCs after NIR-pretreatment might be possible effects of this radiation on the progression through the cell cycle: NIR is described to stimulate proliferation (3,4). If now—as a consequence of NIR treatment—more cells would become X-irradiated during S-phase or mitosis, genetic instability could be stimulated, due to higher sensitivity of these cells against ionizing radiation (49). Again, this possibility could be ruled out by our analysis of cell cycle progression, showing no differences at the time point of X-irradiation, as indicated by FACS analysis and determination of mitotic indices. Remarkably, although NIR showed no impact on the cell cycle progression at the time of irradiation, it revealed strong alterations in the following 24 h: similar to X-radiation, NIR alone significantly decreased BrdU uptake when compared to untreated controls. This is surprising, since—as noted above—it is mostly described as a proliferation-stimulating agent (6,30,50–52).

However, according to a review of Piazena and Kelleher, there is only one publication dealing with NIR effects on proliferation, based on a NIR treatment that is comparable to our experimental setup regarding the used NIR dose and wavelength: in this case the authors described an increase in the mitotic index after a dose of 810 kJ m−2, which is still above the highest dose used in our dose–response experiments (720 kJ m−2) (30,53). Experiments with higher doses were not carried out, since due to our experimental setup, higher doses than 720 kJ m−2 would end up in more than 90 min exposure to serum starvation, room temperature and NIR-induced ROS production. As noted by Schroeder et al., such long periods might lead to compensatory cellular responses, probably ending up in a covering of the essential NIR effects (54).

In contrast to the NIR-based proliferation effect, the proliferation decrease after ionizing X-radiation can be explained by the induction of several damages including DNA DSBs, leading to cell cycle arrest, guaranteeing genomic stability and preventing cells from undergoing malignant transformation (55–59). Against this background it is amazing to see that after the combination of two proliferation-decreasing treatments, more cells enter S-phase when compared to the exclusive X-ray exposure (see Fig. 6). This is a strong hint for an impairment of cell cycle checkpoints after NIR pretreatment. Taken for its own, this might be a positive effect regarding tumor therapy: As noted above, the induction of cell cycle checkpoints is an important feature for maintaining genomic stability after irradiation (55–59). Therefore, the disturbance of this mechanism by NIR might lead to higher ratios of cellular death after tumor irradiation, due to an increase of genomic instability. This is particularly noteworthy, since the improper S-phase entry might even happen in the presence of higher DNA DSBs numbers (see Fig. 7), which increases the probability of an improper replication of genomic material. Talking about higher numbers of DNA DSBs, we interpreted this result as an impairment of DNA DSBs repair in NIR-pretreated X-irradiated cells, since we observed neither an induction of DNA DSBs by exclusive NIR treatment, nor higher numbers of directly induced DNA DSBs in the double-treated cells. Instead, higher γH2AX-foci-numbers (see Fig. 7a) and elevated γH2AX-levels (see Fig. 7c) could only be found after several hours of repair. A possible reason for this slower repair might be found in the higher amount of 8-oxo-dG-adducts that emerge within NIR-treated cells. Since the ratio of single- to DSBs produced by ROS is on the order of 10 000:1 (60), the elevated ROS level in NIR-pretreated cells might bind specific proteins involved in base excision repair and DNA DSBs repair, at the site of DNA single-strand breaks. Thereby, NIR treatment might reduce DNA DSBs-repair capacity. Possible candidates for these proteins are XRCC1, PARP-1 and polynucleotide kinase, which all have been described to be involved in DNA single-strand break repair and DNA DSBs repair by non homologous endjoining (61–65). However, talking about elevated MC levels, it is implausible that a moderate increase in resisting DNA DSBs should be the only reason for this striking phenomenon, notably since MC still emerges when all the induced DNA DSBs should already be repaired (see Fig. 2d–e). Therefore, it is interesting to see that photodynamic therapy in combination with the ROS-producing photosensitizer Zn(II)-phthalocyanine (ZnPc) was also described to produce elevated MC levels, due to altered configurations of the mitotic spindle (66). So in both cases, the combination of light with a ROS-producing treatment (X-radiation and ZnPc) triggers the development of aneuploidy. Thus, our data are in accordance with the results of Chae et al., claiming oxidative stress as an inductor of multipolar spindle formation and centrosome hyperamplification (67). Taken together, our data indicate that the preconditions for the preferential emergence of MCs are fulfilled after NIR pretreatment: due to impaired cell cycle checkpoints, more cells enter S-phase in presence of higher DNA DSBs numbers as well as higher ROS levels, probably ending up in the formation of multipolar spindles and centrosome hyperamplification and—finally—MC (67,68).

ROS as a reason for improper cell cycle regulation

Our results indicate an increase in genomic instability of X-irradiated cells after NIR pretreatment, as a consequence of improper cell cycle regulation, more precisely as an overriding of the G1/S-checkpoint. Since NIR is known to interact with mitochondria, which—in turn—might regulate cellular proliferation by ROS-signaling (1,3), we analyzed the level of intracellular ROS in differently treated cells. Our results show that an exclusive NIR treatment increases the 8-oxo-dG level, as a major product of DNA-oxidation by ROS, when compared to the untreated controls. Furthermore, higher amounts of ROS could be detected in NIR-pretreated cells after ionizing radiation, when compared to exclusively X-irradiated cells (see Fig. 5). A possible explanation for these higher ROS levels might be the significant alterations within the mitochondrial membrane potential independent MitoTracker Green® fluorescence signal 24 h after NIR treatment (see Fig. 4). This result indicates either an increase in mitochondrial mass (39,41) or changes within the intracellular Redox-status, since MTG-fluorescence is also altered by H2O2 (40). Interestingly, the two (3T3 fibroblasts and R28 progenitors) of three tested cell lines that showed this alteration were more susceptible for MC formation after low X-ray doses, when compared to unaltered keratinocytes (HaCaT, see Fig. 2). So in the end, NIR-induced changes within mitochondrial status might be responsible for higher intracellular ROS levels after X-irradiation for the following reason: Analyzing the impact of mitochondria on genetic stability, two studies could show an increase of genetic instability in cells with an elevated mitochondrial mass after irradiation (20,69). This increase was—in both cases—accompanied by a decrease in mitochondrial membrane potential, indicating an impaired mitochondrial functionality that might lead to higher ROS steady state levels in the unstable cells (20,69). These elevated ROS levels might now be able to regulate gene expression, mostly of genes that are associated with proliferation (4) via the mitochondrial retrograde signaling pathway (3), probably leading to MC formation. However, since we could not observe a higher mitochondrial mass within HaCaT cells after NIR (see Fig. 4c), there might be additional mechanisms involved in MC formation, independent from the mitochondrial status. So in summary, we hypothesize that the increase of cells that override the X-irradiation-induced G1/S-checkpoint, is due to an imbalance of cell cycle checkpoint and/or proliferation triggering proteins, induced—amongst others—by the NIR-caused changes within the metabolic status. Thus, our data support recent data, claiming that the probability of irradiated cells to undergo MC depends on their metabolic state (20,21,70–72).

Finally we can conclude that our studies revealed a significant increase in genomic instability, probably due to a changed metabolic state after the combination of physiological doses of NIR and clinically relevant X-radiation doses. These findings might be of clinical relevance, because on one hand, the impaired DNA-damage repair and the increased ROS levels as well as the increased proliferation might be expedient for the elimination of tumor cells. On the other hand, the combination of both radiation qualities promoted MCs, possibly allowing tumor cells to gain resistance to cancer therapy or healthy tissue to amass genomic instability, respectively. So in the end, we can conclude that exposition of patients to NIR during cancer therapy needs more attention in future cancer research.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Acknowledgements— We thank Prof. Dr. M. Löbrich for helpful discussions and kindly providing microscope capacity. The antibody developed by S. J. Kaufmann (BrdU) was obtained from the Developmental Studies Hybridoma Bank Department of Biological Sciences, University of Iowa, Iowa City, USA.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Figure S1. NIR irradiation does not enhance heat shock protein expression. RT-PCR of mRNA for Hsp70, Hsp27 and GAPDH were performed for NIH/3T3 fibroblasts in sham-exposed controls (sham) and 24 h (left panel) as well as 48 h (right panel) after irradiation with 360 kJ/sqm NIR (+NIR). No enhanced expression levels were observable. Bands are representative for three independent experiments (n = 3).

Figure S2. NIR irradiation does decrease proliferation in R28 retinal precursor cells in a dose-dependent manner. Cell proliferation was detected by BrdU-uptake 24–48 h after irradiation with 0, 180, 360 and 720 kJ/sqm NIR. Data are represented as means (n = 2) ± SE (***P < 0.001).

FilenameFormatSizeDescription
PHP_1031_sm_FigS1.tif3499KSupporting info item
PHP_1031_sm_FigS2.tif1633KSupporting info item

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