|Year : 2011 | Volume
| Issue : 2 | Page : 89-95
Analysis of gamma induced DNA strand breaks and repair in normal HACAT and tumor HT 29 cells using comet assay
Praveen Joseph1, NN Bhat2, Narayana Yerol1
1 Department of Studies in Physics, Mangalore University, Mangalagangotri, India
2 Department of Studies in Physics, RPAD, Bhabha Atomic Research Center, Mumbai, India
|Date of Web Publication||12-Jul-2012|
Department of Studies in Physics, Mangalore University, Mangalagangotri
Source of Support: None, Conflict of Interest: None
Ionizing radiation is a potent inducer of DNA damage and comet assay provides a very sensitive method for detecting strand breaks and measuring repair kinetics in single cells. The DNA repair capability influence cell sensitivity to ionizing radiation and predicting radio sensitivity of a cell population is very important for radiation therapy. In the present study the effect of gamma radiation on inducing DNA damage in two cell lines, normal human keratinocytes (HaCaT) and human tumor adenocarcinoma (HT 29) has been analyzed. Radiation treatment has been carried out using gamma radiation from a 60 Co source and the induction and repair of DNA strand breaks were quantified using alkaline comet assay. Repair studies were performed by incubating the sample at 37 0 C for different recovery times with the aim of elucidating repair kinetics. The relation between initial DNA strand breaks and the rejoining kinetics of the strand breaks has been studied in the present investigation. The average repair half time for both the cell lines has been found out. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose and repair time. It is observed that a comparatively radio resistant tumor cell line HT 29 has a shorter repair half time compared to that of normal cell HaCaT. Both the cell lines showed a dose-dependent repair activity and it indicates that the repair rate is proportional to the induced damage.
Keywords: Comet assay, DNA, gamma radiation, HaCaT, HT 29, repair kinetics
|How to cite this article:|
Joseph P, Bhat N N, Yerol N. Analysis of gamma induced DNA strand breaks and repair in normal HACAT and tumor HT 29 cells using comet assay. Radiat Prot Environ 2011;34:89-95
|How to cite this URL:|
Joseph P, Bhat N N, Yerol N. Analysis of gamma induced DNA strand breaks and repair in normal HACAT and tumor HT 29 cells using comet assay. Radiat Prot Environ [serial online] 2011 [cited 2020 Aug 6];34:89-95. Available from: http://www.rpe.org.in/text.asp?2011/34/2/89/98393
| 1. Introduction|| |
Ionizing radiation is an interesting physical agent because the deposition of energy caused by its interaction with biological material is a potent inducer of DNA damage due to both free radical formation and direct DNA interactions.  The exposure of cells to ionizing radiation results in induction of diverse types of DNA damages and of repair processes. , Another aspect that increases interest in ionizing radiation is its use in the treatment of cancer patients. The radiation sensitivity of tumor as well as healthy tissues is crucial for successful cancer radiotherapy. Since the vital target for radiation damage is not the whole body but individual cells or DNA to be more specific,  the tissue response to radiotherapy may depend on the DNA repair potential of the cells.  The DNA repair capability influence cell sensitivity to ionizing radiation and predicting radio sensitivity of a cell population is very important for radiation therapy. 
Over the past decade, the comet assay has become one of the standard methods for assessing DNA damage, with applications in DNA damage and repair. The assay attracts adherents by its simplicity, sensitivity, versatility, speed, and economy.  It provides a very sensitive method for detecting strand breaks and measuring repair kinetics in single cells.  The alkaline method of comet assay enables detection of the broadest spectrum of DNA damage and can detect double and single strand breaks and alkali-labile sites.  In heterogeneous cells or tissues, average responses can mask the presence of a small number of radiation-sensitive cell. For tumors, this distinction is critical since normal diploid cells typically contaminate the sample and can prevent an accurate estimate of tumor cell response to treatment. Exposure to ionizing radiation produces a random distribution of DNA damages within a population of irradiated cells,  and this heterogeneity can be calculated using comet assay.
In the present study, the effect of gamma radiation on human tumor adenocarcinoma (HT 29) and normal human keratinocytes (HaCaT) cells has been analyzed. The induction and repair of DNA strand breaks after treatment with 60 Co radiation were studied using alkaline comet assay. Repair studies were performed for different recovery times with the aim of elucidating repair kinetics. The cell population heterogeneity both in DNA damage and in rejoining activity was examined. A comparative study over the differences in radiation induced DNA damage and its repair in HT 29 and HaCaT cells has been carried out.
| 2. Materials and Methods|| |
2.1. Sample preparation and irradiation
HaCaT and HT 29 cells were grown in 25 ml cell culture dishes (Falcon) and maintained in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal calf serum (FCS; Himedia) at 37°C in a humidified atmosphere containing 5% CO 2 . Cells were passaged every 7 days at a 1:10 split and the experiments were performed on the day of 70-80% confluence. Cultured cells were harvested with 0.025% trypsin EDTA and washed in complete media by centrifugation and diluted to a concentration of 2×10 5 cells/ml. The samples were exposed to gamma radiation using a 60 Co source at Bhabha Atomic Research Centre (BARC), India. The samples were irradiated at room temperature in air and were maintained at 0°C before and after irradiation. For repair kinetics studies cells were incubated at 37°C for different time intervals after radiation exposure. The total dose exposed to the sample varied from 0 to 30 Gy for HT 29 and 0 to 15 Gy for HaCaT cells and the dose rate used was 2 Gy min -1 .
2.2. Comet assay
After irradiation, to quantify the DNA damage the comet assay was carried out under alkaline conditions, by the methods outlined by Singh et al.  100 μl of cell suspension in complete media with a concentration of 2×10 5 cells ml -1 was mixed with 100 μl of low melting point agarose (0.75% in PBS) and layered on frosted slides pre-coated with 200 μl of normal melting point agarose (1.5%). After solidification a layer of 200 μl of low melting point agarose (0.75%) was added to the slides and the slides were placed in a chilled lysing solution containing 2.5 M NaCl, 100 mM Na 2 -EDTA with freshly added 1% Triton X-100 and 10% DMSO, for 12 h at 4°C. The slides were then removed from the lysing solution and washed with alkaline electrophoresis buffer and placed on a horizontal electrophoresis tank filled with freshly prepared alkaline electrophoresis buffer (300 mM NaOH, 1mM Na 2 -EDTA, pH≥13). The slides were equilibrated in the same buffer for 30 min and electrophoresis was carried out at 0.74 V cm -1 , 160 mA for 22 min. After electrophoresis, the slides were washed gently with 0.4 M Tris base buffer (pH 7.2) to remove the alkali and stained with SYBR Green II. Then the slides were visualized at 40X magnification using filter 09 filter set (BP 546/FT580/LP590) using a Carl Zeiss Axioplan microscope with bright field, phase contrast and epi-fluorescence facility (HBO 50 high pressure mercury lamp), 0.5× camera adapter lens, high performance color camera with 750 lines horizontal resolution (KY-F55BE 3CCD, JVC, Japan).
Images of 50 cells per slide were acquired using a digital imaging system and stored. For comparison of DNA damage, the quantitative measurement of the Olive Tail moment (OTM) was carried out using Comet Assay Software Project (CASP).  The total SYBR green fluorescence intensity is taken as total DNA content in the comet. The software allows quantitative measurements of total fluorescence of the comet, fluorescence of the tail, length of migrated DNA fragments and finally calculates the OTM (product of fraction of DNA in the tail and tail length), an internationally most accepted parameter for comparing the DNA damage.
2.3. Statistical analysis
Statistical analysis of the data generated from the comet assay was carried out by importing the result file to the software Microcal Origin Version 8. Data are presented as the mean±standard deviation (S.D.). The mean of the samples at different conditions were compared using the Student's t-test. Differences where P<0.05 were considered to be statistically significant. 
| 3. Results and Discussion|| |
3.1. DNA damage
In the present study, the induction of DNA strand breaks and its repair in normal cells HaCaT and tumor cells HT 29 after gamma irradiation has been analyzed using alkaline comet assay. The optimized and standardized comet assay protocol was used in order to analyze the amount of induced DNA damage in single cells and the DNA repair capacity of different cell lines. [Figure 1] shows images of three cells analyzed with the comet assay. Cell scoring showed no DNA migration in unirradiated cell, which in fact appeared as nucleoids [Figure 1]a. Cells with few strand breaks have a spherical shape, while those with a high frequency of strand breaks present a tail of DNA streaming out from the nucleoid, forming a comet-like appearance. In irradiated cells, DNA fragments migrated out of the head to form the comet tail [Figure 1] b,c thus increasing both comet length and percentage of DNA in the tail. Summary statistics of the amount of DNA in the tail are used as surrogates for the amount of damage and in the present study one particular surrogate, OTM  is used. OTM is the most reliable quantitative indicator of DNA damage, since they refer both to the distance of migration and to the amount of DNA that has migrated from the head region.
|Figure 1: Comet assay image of DNA under normal (1a) and irradiated conditions (1b,c)|
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The dose response characteristic of DNA damage after different doses of gamma irradiation is shown in [Figure 2] and the kinetics of repair are shown in [Figure 3] and [Figure 4]. Each data point in the figure represents a mean value calculated from 50 individual measurements. The 95 % confidence intervals of the mean values are visualized as error bars. From the results shown, the OTM increased steeply as a function of radiation dose in HaCaT [Figure 2]a and HT 29 [Figure 2]b cells and it was possible to quantify the amount of DNA damage accurately. The [Figure 2] is fitted with a linear quadratic equation and it shows that the DNA damage induction is not quite linear with dose in HaCaT and HT 29 cells. In HaCaT cells the variation in OTM with dose is fitted to the equation, OTM = 0.26 + 0.94 * D + (-0.02) * D 2 (R 2 =0.93 and P<0.01). Up to a dose of 10 Gy, it shows a linear increase with dose [Figure 2]a but at higher doses saturation in damage induction can be observed. The variation of OTM with dose in HT 29 cells are fitted to the equation, OTM = 0.71+0.42 * D + (-0.007) * D 2 (R 2 =0.96 and P<0.0001). In the case of tumor cells HT 29, a linear increase in DNA damage with gamma dose is observed up to a dose of 15 Gy and at higher doses the rate of damage induction tend to decrease.
|Figure 2: a) The induction of DNA damage as measured as Olive Tail Moment (OTM) with increasing gamma dose in HaCaT cells. b) The induction of DNA damage as measured as OTM, with increasing gamma dose in HT 29 cells|
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|Figure 3: a) The variation in Olive Tail Moment (OTM) with repair time for HaCaT cells after irradiation with different doses of gamma radiation. b) The variation in OTM with repair time for HT 29 cells after irradiation with different doses of gamma radiation|
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|Figure 4: a) The variation of Olive Tail Moment (OTM) for HaCaT cells irradiated with different doses gamma radiation and incubated for different time intervals. b) The variation of OTM for HT 29 cells irradiated with different doses gamma radiation and incubated for different time intervals|
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3.2. Rejoining of radiation-induced DNA strand breaks
Limited information is available about the variation in capacity for DNA repair among individuals, which is an important determinant of individual susceptibility to cancer. Comet assay is a suitably robust and sensitive assay to understand the variation in DNA repair in individual cells.  Incubation after radiation exposure allows the repair of induced DNA damage. Rejoining of strand breaks were examined by incubating the cells at 37°C for different time intervals ranging from 0 to 4 h after irradiation with different doses prior to performing the comet assay and it is shown in [Figure 3]a and b for HaCaT and HT 29 cells respectively. The differences in re-joining kinetics for different doses in normal and tumor cells were observed and are plotted in these figures.
From the graphs, it is clear that there is no repair after 2 h of incubation. The maximum repair occurs within one hour after irradiation, and the rate of repair in the first 30 min and 30 to 60 min is almost same showing similar slope in the graph [Figure 3]. Though there is repair at 1 to 2 h of incubation period, the rate of repair and the amount of DNA repaired is comparatively less showing a marked difference in the slope of the graph. In the case of HaCaT cell irradiation with 4 Gy, existing DNA damages after 2 h of repair time is comparable to that of control. But for 6 and 10 Gy doses, unrepaired damages exist after 2 h of incubation which could not be repaired even after 4 h. This indicates that in the case of HaCaT cells, the maximum possible repair of DNA damage occurs immediately within 2 h of irradiation and there is no statistically significant repair after 2 h. The DNA damages present even after 2 h of incubation is residual damages due to that particular dose and cannot be repaired, which is observed only at higher doses such as 10 Gy and was absent at lower doses 4 and 6 Gy. Similarly, in the case of HT 29 cells, a statistically significant level of residual damage observed for 12 Gy irradiation even after 2 h of incubation. At 2 h recovery, the repair of DNA damage is significant for all doses in both the cell lines, however, when cells were allowed to recover for 4 h, residual DNA damage was still observed in the case of irradiation with dose of 10 (HaCaT) and 12 Gy (HT 29).
The dose response of DNA damage measured as OTM for HaCaT and HT 29 cells which are incubated for different time interval ranging from 0 to 4 h is fitted linearly and is shown in the [Figure 4]a and b respectively. While quantifying the DNA damage induction with dose, from the linear plot it is observed that the damage increases linearly with dose with a slope of 0.76 when there is no repair in HaCaT cells ([Figure 4]a, 0 min) (OTM = 0.46 + 0.76 * D (R 2 = 0.92). In the case of tumor cell HT 29, the dose response at lower doses is fitted to OTM = 0.93 + 0.31 * D (R 2 = 0.99, P <0.0001), indicating that the damage increases linearly with dose at lower doses with a slope of 0.31 when there is no incubation for re-joining of strand breaks ([Figure 4]b, 0 min). By comparing these two observations, it is clear that, the repair kinetics is a factor which is much dependent on the initial existing DNA damage. A dose of 10 Gy shows an OTM of 8.79 in HaCaT cells and the OTM obtained for a dose of 12 Gy in HT 29 cells is 4.73. A faster repair rate of damage is observed in the case of HaCaT cells after irradiation with 10 Gy compared to that of HT 29 cells exposed to 12 Gy. In a similar manner, the cells incubated for different time intervals shows a reduction in kinetics of repair with incubation time, since a good amount of damage get repaired by the incubation. It is clear from the plots that the slope of the different graphs decreases with increasing incubation time. That means the kinetics of repair or rate of repair of DNA damage decreases with decrease in initial DNA damage.
The slope of the regression line was calculated for each curve in [Figure 4]a and b and is plotted against repair time in the [Figure 5]a and b respectively for HaCaT and HT 29 cells. The initial decrease in the slope is sharp and, followed by a shallow slope. From these slope- repair curves, the average repair half time of respective cell lines has been calculated. The slope of the regression lines represents the rate of change of OTM with dose after various repair time ranging from 0 to 240 min and is fitted to, Slope = 0.08 + 0.68 exp (- repair time/43.1), (R 2 = 0.99) for HaCaT cells [Figure 5]a and Slope = 0.06 + 0.25 exp (- repair time/31.25) (R 2 =0.99) for HT 29 cells [Figure 5]b. From these equations, the calculated repair half time of HaCaT cells after gamma irradiation is 29. 87 min and that for HT 29 cells are 21. 66 min.
|Figure 5: a) Repair time vs slope for HaCaT cells. The slope is taken from each regression curves of Fig. 4a. b) Repair time vs slope for HT 29 cells. The slope is taken from each regression curves of Fig. 4b|
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The differences in strand break repair is evident by comparing the slopes of the first portions (0- 2 h recovery) of the curves at the three doses [Figure 3], the highest slope and rejoining rate belongs to the highest dose and highest initial damage. After 2 h recovery, a similar value of OTM is attained for all doses and the second portion (2 - 4 h) of each curve has approximately the same slope regardless of dose. The slopes of the regression line were calculated for each curve in [Figure 4]a and b, and are plotted against OTM in the [Figure 6]a and b respectively. The [Figure 6]a for HaCaT cell is fitted linearly to, slope = (-0.09) + 0.1 * OTM (R 2 = 0.99, P<0.0001) and that of HT 29 cells is, Slope = (-0.09) + 0.08 * OTM (R  =0.99, P<.001) [Figure 6]b. It is clear from the [Figure 6] that the rate of repair (i.e. the rate change of OTM with repair time) is directly proportional to the amount of damage and the rate increases as OTM increases. The OTM at various re-joining times increases with dose and decreases with the repair time.
Ionizing radiation is a physical agent that primarily targets DNA molecules and produces a vast number of types of damages. The aim of the work was to study the induction and repair of DNA strand breaks in cells after irradiation with different gamma doses. One reason was that the analysis of amount of DNA damage repaired after irradiation with different doses could be the key to fully understanding the seriousness of the damage imparted to the cell system. Comet assay was used to determine that, the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine true DNA strand break re-joining rates.  Using the comet assay, initial re-joining rates after ionizing irradiation were found to be similar for all cells within a population. 
|Figure 6: a) Olive Tail Moment (OTM) vs slope for HaCaT cells (slope obtained from the regression curves of Fig. 4a). b) OTM vs slope for HT 29 cells (slope obtained from the regression curves of Fig. 4b)|
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By comparing DNA repair kinetics of one normal and tumor cell lines, a broad spectrum of different DNA repair capacities could be observed. Previous studies have shown that initial radiation-induced DNA damage is the same in both radio resistant and radiosensitive variants of cell lines.  But in the present study, in normal cell HaCaT and tumor cell HT 29, shows that the initial DNA damage induction for the same dose varies with the cell sensitivity to radiation. The comparison of the two different cell lines in the rejoining process is in agreement with previous results, , and they showed a dose-dependent repair activity. The dose-dependent repair activity may not indicate that a certain amount of damage is needed to activate the repair process but the repair rate is proportional to the induced damage. Comet assay that examines each individual cell also provides the means to identify the heterogeneity of DNA damage. Cell response to ionizing radiation showed increased heterogeneity in DNA damage measured as OTM at higher doses. The presence of different amounts of damage within the entire population could reflect the stochastic nature of the action of ionizing radiation, but could also be an important method for identifying those cell subpopulations that are either resistant to or sensitive to radiation-induced strand breaks. 
| 4. Conclusions|| |
The comet assay has been carried out in HaCaT and HT 29 cells after gamma irradiation in order to understand the behavior of individual cells of varying radiation sensitivity exposed to ionizing radiation. Heterogeneity detected using the comet assay can be used to identify the DNA repair deficient cells. Although initial damage induced by ionizing radiation is largely dependent on the radiation sensitivity of the cell line, the repair of DNA damage may be dependent on the amount of damage present at the time. The average repair half time for both the cell lines has been found out from the study and it is observed that the comparatively radio resistant tumor cell line HT 29 has a shorter repair half time compared to that of normal cell HaCaT.
| 5. Acknowledgements|| |
The authors are grateful to Board of Research in Nuclear Sciences (BRNS), Department of Atomic Energy (DAE), Govt. of India, for the financial support. The help received from Dr. Y. S. Mayya, Dr. B. K. Sapra, Dr. B. Sreedevi and Dr. K. B. Anjaria from RPAD, Bhabha Atomic Research Center (BARC), India, is thankfully acknowledged.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]