Mutagenesis Advance Access published online on February 5, 2008
Mutagenesis, doi:10.1093/mutage/gem054
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Potential use of the comet assay in the clinical management of cancer
Cancer & Ageing Research Group, Biomedical Sciences Research Institute, University of Ulster, Northern Ireland, Coleraine BT52 1SA, UK
The comet assay has been widely used to measure a range of cellular responses to DNA damage and has found applications in genotoxicity studies, bio-monitoring, ecological testing and in the study of human disease. This review discusses how the comet assay has been applied to the study of DNA damage and repair associated with cancer. The potential of the assay as a tool for predicting an individual's tumour sensitivity to radiation and to various chemotherapeutic drugs is examined, as well as outlining the usefulness of the assay in assessing oxidative stress within tumours. In addition, we review the use of the comet assay in investigations of the DNA-damaging effect of anti-neoplastic drugs and radiation used during cancer therapy. The advantages and limitations of the comet assay in carrying out all these studies are outlined, and the suitability of the comet assay for use in the clinical management of cancer is discussed.
 |
Introduction |
|---|
 Top Introduction Use of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
The comet assay is now widely accepted as a standard method for assessing DNA damage in individual cells. It has been used in a broad variety of applications including human bio-monitoring, genotoxicology, ecological monitoring and as a tool to investigate DNA damage and repair in different cell types in response to a range of DNA-damaging agents (1
). In addition, many studies have successfully demonstrated how the comet assay can be applied to the analysis of cells derived from a variety of different human tissues, lending considerable weight to the suggestion that this assay could benefit many areas of clinical investigation by providing valuable information about the intrinsic DNA characteristics of individual cells and their responses to various external factors, such as radiation, chemicals and drugs. This type of information would prove particularly relevant in the diagnosis, prognosis and treatment of cancer. Readily available accurate and informative data are fundamental to the successful management of tumours with the ultimate aim of achieving a positive outcome for the patient. From a practical point of view, the comet assay would be eminently suitable for use in a clinical setting since it is a relatively simple and inexpensive technique, which requires only a few cells and results can be obtained within a matter of hours. However, despite these advantages, it has not been widely employed as a standard analytical tool within clinical laboratories, partly because there is still some doubt about its reliability, reproducibility and validity. Hence, in this paper, we focus on the applications of the comet assay in the study of cancer, reviewing studies that have investigated DNA damage and repair, mutagen sensitivity and biomarker potential in human tumour cells to help determine whether the comet assay really does have promise for clinical testing for cancer and what aspects of the assay need to be addressed in order to provide convincing reassurances about its potential worth and usefulness.
|
Use of the comet assay in cancer studies |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
DNA damage and defective DNA repair are the underlying molecular events driving the initiation and progression of cancer. It is therefore not surprising that many studies have used the comet assay to investigate DNA damage and repair characteristics in a wide range of tumour cells in response to a variety of DNA-damaging agents. These studies include both investigations on human tumour cell lines and on tumour cells extracted from cancer patients. Table I demonstrates the range of cancers and end points that have been assessed to date in cells extracted from clinical biopsy material.
|
Many of these studies show that results generated by the comet assay provide important information about the nature of the particular cancer, which could be used by oncologists to inform them as to the best possible course of intervention. As discussed below, these studies, and others, provide a practical solution to some of the problems encountered in the treatment of cancer.
|
Prediction of tumour radiosensitivity |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
The ability to predict the radiosensitivity of individual tumours has long been considered the ‘holy grail’ of radiation biology (18
). A number of different factors will be considered in deciding upon a therapeutic radiation regimen for a cancer patient, but unfortunately there is still no definitive way of predicting whether the tumour will respond to radiotherapy. Tumour response to treatment will inevitably vary due to a range of factors including the type of tumour being treated, as well as the specific genetic make-up of the individual patient (19). At the cellular level, whether or not a tumour cell will die depends on factors such as the type of radiation used, cell-cycle position, oxygen level within the cell and the expression of various oncogenes and growth factors (20). With so many variables capable of exerting an influence on response to radiotherapy, it is not surprising that a wide variability in patient response exists. Therefore, it seems clear that a clinical test for accurately predicting tumour response to radiation on an individual basis would be a valuable asset in the cancer clinic.
A number of different approaches have attempted to predict the likely response of individual tumours to radiation. Currently, much work is focused on the use of microarray technology as a way to generate genetic signature profiles which will indicate the probable response of a tumour to radiation [reviewed in (21)]; this approach represents promising and revolutionary technology. However, the implementation of this technology in predictive radiation biology has yet to be validated and there are still some important limitations with regards to interpretation of gene expression data, complications raised by cell heterogeneity and expense. Moreover, assays that measure phenotypic characteristics of cells (such as DNA repair) are still necessary to validate and substantiate the findings of such technology.
Previously, a more widely used assay for predictive tumour testing was the clonogenic cell survival assay (22). In particular, the SF2 clonogenic assay, which measures the surviving fraction of tumour cells after 2 Gy X-ray irradiation, was regarded as an accurate method for measuring and predicting tumour cell radiosensitivity at clinically relevant doses (23,24). However, the main disadvantages of this assay are that a number of weeks are required to obtain a result, and many excised tumours fail to grow in soft agar (25). These limitations have stimulated an interest in developing methods that might provide a quicker, and more robust, measure of tumour radiosensitivity so that results could be considered in treatment planning.
Such methods include measuring the potential doubling time (Tpot) of the tumour (26), tumour hypoxia measured by pO2 (27), the percentage of apoptotic or viable cells (28) and the index of thymidine and BudR labelling (29). In addition in a range of tumour types, the immunohistochemical detection of many specific proteins, e.g. p53, p21 and EGFR, have been evaluated as possible indicators of tumour cell radiosensitivity (30–32). Functional cell imaging by means of positron emission tomography or single-photon emission computerized tomography has also been employed to measure tumour cell proliferation, metabolism, hypoxia and apoptosis in situ in response to radiation (33,34). All these techniques show promise, but for a variety of reasons are not yet routinely implemented in clinical practice.
The comet assay is another possible predictive test and offers some distinct advantages in comparison to the techniques outlined above. It is simple and quick to do, relatively inexpensive and it does not require clonogenic cell growth as it is carried out on single-cell suspensions, all criteria which would make it appropriate for routine testing in a clinical context. Initially the neutral comet assay was used to assess radiosensitivity in a variety of tumour cell lines (35–37). However, the radiation doses required to produce measurable levels of double strand breaks (typically 20–150 Gy) were far greater than those used clinically, which also made comparison with the SF2 clonogenic assay difficult. The alkaline comet assay, however, allows radiation effects to be measured at low, clinically relevant, doses (0–6 Gy). The alkaline comet assay has been successfully used to demonstrate that radiosensitivity can be measured in a range of tumour cell lines including cervical (38,39), colon (40,41), bladder (25,42–44), prostate (40,45), renal (46), breast (47), lung (48) and ovarian (49). Since each cell line in effect represents a different individual, these studies collectively demonstrate that the comet assay is capable of detecting intrinsic differences in radiosensitivity between different tumour cell lines.
However, to be accepted as a routine test suitable for clinical use, the alkaline comet assay needs to be validated against other methods of measuring radiosensitivity and also must prove applicable to tumour cell samples extracted from cancer patients. To address the first of these issues, several studies have compared radiosensitivity measured by the alkaline comet assay and SF2 clonogenic survival in various cell lines. Three independent studies published concurrently in 2003 demonstrated a strong correlation (all R2 values > 0.95) between the two methods in measuring the radiosensitivity of several bladder (25,42) and colorectal (40) tumour cell lines after irradiation with <10 Gy in vitro, indicating that both initial DNA damage and residual damage can be used to predict for radiosensitivity in these cell lines. Similar studies have also shown a correlation between radiosensitivity measured by the comet assay and SF2 clonogenic survival following in vitro radiation of human melanoma (50), cervical (39) and ovarian (49) tumour cell lines. In normal fibroblasts, initial DNA damage, but not DNA repair, has also been shown to correlate with clonogenic survival (51). Although more concerted investigation in this area would allow more definitive conclusions to be made, the results of these studies would suggest that the alkaline comet assay may indeed prove to be a reliable and comparable alternative to the time-consuming clonogenic survival assay.
When cells have been extracted from excised human tumours, the alkaline comet assay has successfully identified variations in radiosensitivity in studies of bladder (42), head and neck (14), breast (6) and a range of metastatic tumours (52). These studies further demonstrate how the comet assay could support clinical decision making. Just a few cells extracted from a biopsy can be quickly assessed for their response to radiation, thereby giving additional diagnostic information to the more standard cytological techniques. A further, related approach involves investigating the extent of DNA damage and repair ability in response to radiation is measured in surrogate cells, such as lymphocytes, extracted from cancer patients (see below).
|
Prediction of tumour chemosensitivity |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
A similar problem in predicting tumour response occurs when the cancer therapy involves a chemotherapeutic drug. Drug resistance is a major obstacle to successful chemotherapy and it is not yet possible to predict the intrinsic resistance of tumour cells to a given drug or to forecast the ability of the tumour cells to develop resistance over a period of time (53
). Here too, the comet assay offers a versatile candidate for a clinical assay since modifications to the standard comet assay allow the measurement of DNA cross-linking and alkylation rather than DNA strand breakage (54,55). Naturally, there are some limitations to this approach in that not all drugs produce the sort of damage that can be measured by the comet assay, but it has still proven a useful tool in assessing the sensitivity of cancer cells to particular compounds.
As with radiation, the sensitivity of cells to chemical mutagens, as well as their ability to subsequently repair the damage, can be measured using the comet assay. Several drugs, which are routinely used in cancer chemotherapy, have been investigated in a range of human cancer cell lines in vitro, including cisplatin (56,57), mitomycin C (43,58), tamoxifen (59,60) and epirubicin (61). The success of this approach means that investigators can explore if tumour cells can be further sensitized to the action of chemotherapeutic drugs by the introduction of other factors. For example, cells can be treated in vitro with combinations of drugs (61,62), with compounds that interfere with the DNA repair pathways (63) or with a combination of drugs and photo damaging agents (64,65). Thus, comet assay analysis can assess if increased DNA damage and/or tumour cell death occurs. These in vitro studies on human cancer cell lines demonstrate the value of the comet assay as a tool for quickly establishing conditions in which drugs work most effectively both as single agents and in various combinations.
With this in mind, it would seem natural to apply similar techniques to tumour cells isolated from biopsy material obtained from individual cancer patients. However, few studies have taken this approach. Instead, the more common approach involves indirect measurement of the effect of anti-neoplastic drugs by assessing DNA damage in surrogate cells, such as peripheral blood lymphocytes (PBL), from cancer patients undergoing chemotherapy. This allows for easier sample collection, but is not without its problems as discussed below.
|
Measuring DNA damage in PBL of cancer patients |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
The comet assay is now a widely used tool in the field of genetic toxicology testing on human, animal and plant cells in vitro and in vivo (66
). In human studies of this type, the majority of investigations examine PBL for increased levels of DNA damage, although urothelial cells extracted from urine samples and buccal cells can also serve as suitable surrogate cells. Collection of PBL is usually less invasive than target tissue and they are the surrogate cells of choice in studies where target tissue is not readily attainable (66). Hence, the comet assay has been used in assessing the exposure (often occupational) of a population to various mutagenic compounds (67). With specific regard to cancer, this approach has been used experimentally to measure the levels of basal DNA damage in PBL of cancer patients, as well as assessing if the cells are more susceptible to the DNA-damaging effects of radiotherapy or chemotherapy.
Several studies have shown that basal DNA damage is indeed increased in PBL of patients suffering from a variety of different cancers, including head and neck (68), breast (69–71), renal (72), oesophageal (2), bladder (73), ovarian (3) and lung (74). Many of these studies and others have also extracted PBL from cancer patients (usually prior to radiotherapy or chemotherapy) and exposed them to DNA-damaging agents in vitro to assess if susceptibility to DNA damage and subsequent repair capacity is significantly different from control samples (Table II).
|
With respect to radiation, several studies have demonstrated higher initial and/or residual DNA damage and lower repair rate following irradiation of PBL in vitro as compared to controls (68
,69,74–76). Likewise, several studies have shown similar increases in DNA damage and decreases in repair efficiency in response to in vitro treatment with chemical mutagens such as doxorubicin (77), N-methyl N-nitro N-nitrosoguanidine (70), bleomycin (78,79) and benzo(a)pyrene diolepoxide (72). Furthermore, many other studies have examined PBL of cancer patients who are undergoing chemotherapy or radiotherapy to see if the effect of radiation or anti-neoplastic drugs results in lymphocyte DNA damage in vivo; this approach is used as a surrogate indicator of how the tumour cells may be affected (Table III). These studies have shown that DNA damage is increased and/or DNA repair capacity is decreased in PBL samples from patients with a variety of cancers.
|
In evaluating these studies, the critical issue is the judicious interpretation of the data generated by the comet assay. It must not be forgotten that PBL are surrogate cells and therefore damage detected in these cells may not necessarily reflect the damage in tumour cells or any other target tissue, although it is worth noting that some studies, using methods other than the comet assay, have shown good correlation between patient response to chemotherapy and DNA adducts (89
,90) and DNA repair capacity (91) in blood cells. Also, as most of the studies above acknowledge, basal and residual levels of DNA damage in PBL can be influenced by many external factors, such as smoking, diet, medication, air pollution and exercise among others, resulting in a wide intra-individual and inter-individual variability (67). Such a range of confounding factors has the effect of undermining the worth and appeal of the comet assay as a predictive clinical test. Thus, it seems clear that until a standard protocol is agreed upon and multi-laboratory validation trials are carried out for quality control, it will be difficult for the comet assay to gain widespread clinical acceptance as a reliable method for measuring DNA damage in PBL caused by exposure to radiotherapy or anti-neoplastic drugs (67,92). |
Measurement of tumour hypoxia |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
Tumour hypoxia has been linked to poor tumour response to both radiotherapy and chemotherapy and is also associated with a poor outcome following surgery (93
). The ability to measure hypoxia in tumours can provide important predictive information about probable tumour response, thereby allowing the individualizing of treatment according to hypoxic status (94). Currently, one of the most common methods for measuring tumour hypoxia is by using an Eppendorf oxygen microelectrode to measure pO2 levels in solid tumours (95). However, this approach has difficulty in distinguishing between hypoxic and necrotic regions and is limited to accessible tumours (93). The comet assay can be used to measure tumour hypoxia on any excised tumour biopsy, based on the premise that radiation produces approximately three times more DNA damage in well-oxygenated as compared to hypoxic cells (6,15). Cells can be obtained by fine-needle aspirate biopsy following in vivo irradiation of the tumour and the assay is not affected by necrosis. This approach has been used to successfully measure tumour hypoxia in radiotherapy patients with head and neck tumours (13), breast (6) and a range of metastatic tumours (17,52). However, there are some limitations in that radiation doses in excess of 3.5 Gy are required immediately before the biopsy in order to detect hypoxic cells with the comet assay and there is a risk of contamination from circulating white blood cells (52).
While both the oxygen microelectrode and comet assay techniques are capable of identifying hypoxic tumours, studies comparing the two techniques in human tumours have failed to show a consistent correlation (13,16,17,52), which presumably reflects the intrinsic differences in how they measure hypoxia. Although there is a recognition that hypoxia is a significant factor affecting both tumour response to treatment and patient survival, there is currently no consensus as to the best way to measure hypoxia (94). The comet assay offers one method to address this issue but the results are unlikely to be accepted unless corroborated with other methods of hypoxic cell determination.
|
Clinical applicability of the comet assay |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
A recent review by Taube et al. (96
) states that a new diagnostic tool must pass three major tests before it is adopted for routine clinical use: (i) it must be robust and reproducible, (ii) the clinical value to the patient must be proven and (iii) the clinical community must be convinced of the value and benefit of such a tool. These pertinent points should act as a guideline in focusing our collective efforts in seeking clinical acceptance for the comet assay as a diagnostic technique and predictive test for response to radiotherapy/chemotherapy in the management of cancer and for its application to the management of other disorders. Indeed, these points also apply to other potential methods for predictive assays and a series of stringent guidelines has already been produced for tumour marker prognostic studies to help minimize poor study design, non-standardized assays that lack reproducibility and inappropriate or misleading statistical analyses (97). A similar list of guidelines for comet assay studies would help to encourage more transparent and thorough investigations, thereby allowing relevant and unambiguous information from each study to be available to others in a way that will inevitably improve the quality, significance and applicability of comet assay data and results.
In the meantime, it may prove fruitful to make a concerted effort to qualify and collate all existing published comet data into a central database allowing pooled analysis of previous work. Such an approach has successfully resulted in establishing that high chromosome aberration frequency in PBL was a reliable predictive biomarker for cancer incidence, a conclusion that was based on the work of a coordinated, international, multi-centre cohort study which analysed a joint database comprised of data on 3184 subjects from previous studies (98). A corresponding approach for comet assay studies might also yield similarly pertinent information. Indeed, it is worth noting that an inter-laboratory consortium has already been established in an attempt to implement standard, reliable protocols (including the comet assay) for sample preparation and analysis of low levels of 8-oxo-7,8-dihydro-2'deoxyguanosine in lymphocyte DNA (99). With such collaboration and teamwork already in place, an excellent opportunity exists to establish a separate inter-laboratory committee focused on the comet assay and its potential in the clinical management of cancer. In addition, efforts must be made wherever possible to forge and strengthen collaborations with colleagues in hospitals and clinical laboratories, not just with the aim of obtaining useful cell samples, but with a view to establishing the worth of the comet assay in the eyes of the clinical community. Without support and backing from oncologists, clinicians and regulatory authorities, the comet assay will struggle to gain acceptance as a routine diagnostic tool in cancer management.
|
Conclusions |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
The studies discussed in this paper demonstrate that the comet assay can successfully measure DNA damage and subsequent repair in a variety of human tumour cells following exposure to both radiation and mutagenic compounds. It is a sensitive, rapid and versatile technique, lending itself well to the study of a range of DNA-damaging agents, such as those used in cancer therapy. However, despite this, the comet assay will not, and indeed should not, be used in the management of cancer or any other disease unless fundamental issues relating to experimental validation, standardization and data interpretation are addressed.
|
Acknowledgments |
|---|
Conflict of interest statement: None declared.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +44 28 70323285; Fax: +44 28 70324375; Email: dj.mckenna{at}ulster.ac.uk
|
References |
|---|
|
TopIntroductionUse of the comet...Prediction of tumour...Prediction of tumour...Measuring DNA damage in...Measurement of tumour hypoxiaClinical applicability of the...ConclusionsReferences |
|---|
-
1. Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol. Biotechnol. (2004) 26:249–261.[CrossRef][Web of Science][Medline]
2. Olliver JR, Hardie LJ, Gong Y, Dexter S, Chalmers D, Harris KM, Wild CP. Risk factors, DNA damage, and disease progression in Barrett's esophagus. Cancer Epidemiol. Biomarkers. Prev. (2005) 14:620–625.
3. Baltaci V, Kayikcioglu F, Alpas I, Zeyneloglu H, Haberal A. Sister chromatid exchange rate and alkaline comet assay scores in patients with ovarian cancer. Gynecol. Oncol. (2002) 84:62–66.[CrossRef][Web of Science][Medline]
4. McGlynn AP, Wasson GR, O'Reilly S, et al. Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay. Br. J. Cancer (2003) 88:895–901.[CrossRef][Web of Science][Medline]
5. Johnstone EC, Lind MJ, Griffin MJ, Boddy AV. Ifosfamide metabolism and DNA damage in tumour and peripheral blood lymphocytes of breast cancer patients. Cancer Chemother. Pharmacol. (2000) 46:433–441.[CrossRef][Web of Science][Medline]
6. Olive PL, Durand RE, Le Riche J, Olivotto IA, Jackson SM. Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers. Cancer Res. (1993) 53:733–736.
7. Olive PL, Trotter T, Banath JP, Jackson SM, Le Riche J. Heterogeneity in human tumour hypoxic fraction using the comet assay. Br. J. Cancer Suppl. (1996) 27:S191–S195.[Medline]
8. Zhang H, Spitz MR, Tomlinson GE, Schabath MB, Minna JD, Wu X. Modification of lung cancer susceptibility by green tea extract as measured by the comet assay. Cancer Detect. Prev. (2002) 26:411–418.[CrossRef][Web of Science][Medline]
9. Sanchez P, Penarroja R, Gallegos F, Bravo JL, Rojas E, Benitez-Bribiesca L. DNA damage in peripheral lymphocytes of untreated breast cancer patients. Arch. Med. Res. (2004) 35:480–483.[CrossRef][Web of Science][Medline]
10. Castellon E, Clementi M, Hitschfeld C, Sanchez C, Benitez D, Saenz L, Contreras H, Huidobro C. Effect of leuprolide and cetrorelix on cell growth, apoptosis, and GnRH receptor expression in primary cell cultures from human prostate carcinoma. Cancer Invest. (2006) 24:261–268.[CrossRef][Web of Science][Medline]
11. Eastham AM, Marples B, Kiltie AE, Orton CJ, West CM. Fibroblast radiosensitivity measured using the comet DNA-damage assay correlates with clonogenic survival parameters. Br. J. Cancer (1999) 79:1366–1371.[CrossRef][Web of Science][Medline]
12. Kleinsasser NH, Weissacher H, Kastenbauer ER, Dirschedl P, Wallner BC, Harreus UA. Altered genotoxicity in mucosal cells of head and neck cancer patients due to environmental pollutants. Eur. Arch. Otorhinolaryngol. (2000) 257:337–342.[CrossRef][Medline]
13. Le QT, Kovacs MS, Dorie MJ, et al. Comparison of the comet assay and the oxygen microelectrode for measuring tumor oxygenation in head-and-neck cancer patients. Int. J. Radiat. Oncol. Biol. Phys. (2003) 56:375–383.[CrossRef][Web of Science][Medline]
14. Terris DJ, Ho EY, Ibrahim HZ, Dorie MJ, Kovacs MS, Le QT, Koong AC, Pinto HA, Brown JM. Estimating DNA repair by sequential evaluation of head and neck tumor radiation sensitivity using the comet assay. Arch. Otolaryngol. Head Neck Surg. (2002) 128:698–702.
15. McLaren DB, Pickles T, Thomson T, Olive PL. Impact of nicotinamide on human tumour hypoxic fraction measured using the comet assay. Radiother. Oncol. (1997) 45:175–182.[CrossRef][Web of Science][Medline]
16. Olive PL, Banath JP, Aquino-Parsons C. Measuring hypoxia in solid tumours—is there a gold standard? Acta Oncol. (2001) 40:917–923.[CrossRef][Web of Science][Medline]
17. Aquino-Parsons C, Luo C, Vikse CM, Olive PL. Comparison between the comet assay and the oxygen microelectrode for measurement of tumor hypoxia. Radiother. Oncol. (1999) 51:179–185.[CrossRef][Web of Science][Medline]
18. Peters LJ, Brock WA, Johnson T, Meyn RE, Tofilon PJ, Milas L. Potential methods for predicting tumor radiocurability. Int. J. Radiat. Oncol. Biol. Phys. (1986) 12:459–467.[Web of Science][Medline]
19. Awwad HK. Normal tissue radiosensitivity: prediction on deterministic or stochastic basis? J. Egypt. Natl. Canc. Inst. (2005) 17:221–230.[Medline]
20. Fornace A, Fuks Z, Weichselbaum RR, Milas L. Radiation therapy. In: The Molecular Basis of Cancer—Mendelsohn J, Howley PM, Israel MA, Liotta LA, eds. (2001) Philadelphia, PA: WB Saunders. 423–454.
21. Kruse JJ, Stewart FA. Gene expression arrays as a tool to unravel mechanisms of normal tissue radiation injury and prediction of response. World J. Gastroenterol. (2007) 13:2669–2674.[Web of Science][Medline]
22. Olive PL. The role of DNA single- and double-strand breaks in cell killing by ionizing radiation. Radiat. Res. (1998) 150:S42–S51.[Web of Science][Medline]
23. West CM, Davidson SE, Roberts SA, Hunter RD. The independence of intrinsic radiosensitivity as a prognostic factor for patient response to radiotherapy of carcinoma of the cervix. Br. J. Cancer (1997) 76:1184–1190.[Web of Science][Medline]
24. Bjork-Eriksson T, West C, Karlsson E, Mercke C. Tumor radiosensitivity (SF2) is a prognostic factor for local control in head and neck cancers. Int. J. Radiat. Oncol. Biol. Phys. (2000) 46:13–19.[CrossRef][Web of Science][Medline]
25. McKeown SR, Robson T, Price ME, Ho ET, Hirst DG, McKelvey-Martin VJ. Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation. Br. J. Cancer (2003) 89:2264–2270.[CrossRef][Web of Science][Medline]
26. Alsner J, Hoyer M, Sorensen SB, Overgaard J. Interaction between potential doubling time and TP53 mutation: predicting radiotherapy outcome in squamous cell carcinoma of the head and neck. Int. J. Radiat. Oncol. Biol. Phys. (2001) 49:519–525.[CrossRef][Web of Science][Medline]
27. Nordsmark M, Eriksen JG, Gebski V, Alsner J, Horsman MR, Overgaard J. Differential risk assessments from five hypoxia specific assays: the basis for biologically adapted individualized radiotherapy in advanced head and neck cancer patients. Radiother. Oncol. (2007) 83:389–397.[CrossRef][Web of Science][Medline]
28. Burcombe R, Wilson GD, Dowsett M, Khan I, Richman PI, Daley F, Detre S, Makris A. Evaluation of Ki-67 proliferation and apoptotic index before, during and after neoadjuvant chemotherapy for primary breast cancer. Breast Cancer Res. (2006) 8:R31.[CrossRef][Medline]
29. Tachibana M, Miyakawa A, Nakamura K, Baba S, Murai M, Tazaki H. Role of proliferative activity estimated by bromodeoxyuridine labeling index in determining predictive factors of recurrence in superficial intermediately malignant bladder tumors. J. Urol. (1996) 156:63–69.[CrossRef][Web of Science][Medline]
30. Lopez-Crapez E, Bibeau F, Thezenas S, Ychou M, Simony-Lafontaine J, Thirion A, Azria D, Grenier J, Senesse P. p53 status and response to radiotherapy in rectal cancer: a prospective multilevel analysis. Br. J. Cancer (2005) 92:2114–2121.[CrossRef][Web of Science][Medline]
31. Komuro Y, Watanabe T, Tsurita G, Muto T, Nagawa H. Evaluating the combination of molecular prognostic factors in tumor radiosensitivity in rectal cancer. Hepatogastroenterology (2005) 52:666–671.[Medline]
32. D'Andrea MR, Gasparini G. The future of anti-EGFR therapy. Int. J. Biol. Markers (2007) 22:S88–S93.[Web of Science][Medline]
33. Belkacémi Y, Tsoutsou P, Magné N, Castadot P, Azria D. Metabolic functional imaging for tumor radiosensitivity monitoring. Crit. Rev. Oncol. Hematol. (2007) 62:227–239.[CrossRef][Web of Science][Medline]
34. Van de Wiele C, Lahorte C, Oyen W, Boerman O, Goethals I, Slegers G, Dierckx RA. Nuclear medicine imaging to predict response to radiotherapy: a review. Int. J. Radiat. Oncol. Biol. Phys. (2003) 55:5–15.[CrossRef][Web of Science][Medline]
35. Olive PL, Banath JP, MacPhail HS. Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines. Cancer Res. (1994) 54:3939–3946.
36. Marples B, Longhurst D, Eastham AM, West CM. The ratio of initial/residual DNA damage predicts intrinsic radiosensitivity in seven cervix carcinoma cell lines. Br. J. Cancer (1998) 77:1108–1114.[Web of Science][Medline]
37. Price ME, McKelvey-Martin VJ, Robson T, Hirst DG, McKeown SR. Induction and rejoining of DNA double-strand breaks in bladder tumor cells. Radiat Res. (2000) 153:788–794.[CrossRef][Web of Science][Medline]
38. Gurska S, Farkasova T, Gabelova A. Radiosensitivity of cervical cancer cell lines: the impact of polymorphisms in DNA repair genes. Neoplasma (2007) 54:195–201.[Web of Science][Medline]
39. Banath JP, Macphail SH, Olive PL. Radiation sensitivity, H2AX phosphorylation, and kinetics of repair of DNA strand breaks in irradiated cervical cancer cell lines. Cancer Res. (2004) 64:7144–7149.
40. Dunne AL, Price ME, Mothersill C, McKeown SR, Robson T, Hirst DG. Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells. Br. J. Cancer (2003) 89:2277–2283.[CrossRef][Web of Science][Medline]
41. Price WA, Stobbe CC, Park SJ, Chapman JD. Radiosensitization of tumour cells by cantharidin and some analogues. Int. J. Radiat. Biol. (2004) 80:269–279.[CrossRef][Web of Science][Medline]
42. Moneef MA, Sherwood BT, Bowman KJ, Kockelbergh RC, Symonds RP, Steward WP, Mellon JK, Jones GD. Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity. Br. J. Cancer (2003) 89:2271–2276.[CrossRef][Web of Science][Medline]
43. McKenna DJ, Gallus M, McKeown SR, Downes CS, McKelvey-Martin VJ. Modification of the alkaline comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells. DNA Repair (2003) 2:879–890.[Medline]
44. McKelvey-Martin VJ, Ho ET, McKeown SR, Johnston SR, McCarthy PJ, Rajab NF, Downes CS. Emerging applications of the single cell gel electrophoresis (comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization comets for the identification of damaged and repaired DNA sequences in individual cells. Mutagenesis (1998) 13:1–8.
45. Cardile V, Bellia M, Lombardo L, Scifo C, Renis M. Distinct response to ionizing radiation of human prostate cell lines. Oncol. Rep. (2005) 14:981–985.[Web of Science][Medline]
46. Smyth A, Reid HM, Baker AH, McGlynn H. Modifications of the radiosensitivity of a renal cancer cell line as a consequence of stable TIMP-1 overexpression. Int. J. Radiat. Biol. (2007) 83:13–25.[CrossRef][Web of Science][Medline]
47. Niedbala M, McNamee JP, Raaphorst GP. Response to pulsed dose rate and low dose rate irradiation with and without mild hyperthermia using human breast carcinoma cell lines. Int. J. Hyperthermia (2006) 22:61–75.[CrossRef][Web of Science][Medline]
48. Lee YJ, Sheu TJ, Keng PC. Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin. Biochem. Biophys. Res. Commun. (2005) 335:286–291.[CrossRef][Web of Science][Medline]
49. Bacova G, Hunakova LE, Chorvath M, Boljesikova E, Chorvath B, Sedlak J, Gabelova A. Radiation-induced DNA damage and repair evaluated with ‘comet assay’ in human ovarian carcinoma cell lines with different radiosensitivities. Neoplasma (2000) 47:367–374.[Web of Science][Medline]
50. Wada S, Kurahayashi H, Kobayashi Y, Funayama T, Yamamoto K, Natsuhori M, Ito N. The relationship between cellular radiosensitivity and radiation-induced DNA damage measured by the comet assay. J. Vet. Med. Sci. (2003) 65:471–477.[CrossRef][Web of Science][Medline]
51. Brammer I, Zoller M, Dikomey E. Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts. Int. J. Radiat. Biol. (2001) 77:929–938.[CrossRef][Web of Science][Medline]
52. Olive PL, Durand RE, Jackson SM, et al. The comet assay in clinical practice. Acta Oncol. (1999) 38:839–844.[CrossRef][Web of Science][Medline]
53. Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J. Pathol. (2005) 205:275–292.[CrossRef][Web of Science][Medline]
54. Merk O, Speit G. Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity. Environ. Mol. Mutagen. (1999) 33:167–172.[CrossRef][Web of Science][Medline]
55. Pfuhler S, Wolf HU. Detection of DNA-crosslinking agents with the alkaline comet assay. Environ. Mol. Mutatgen. (1996) 27:196–201.[CrossRef][Web of Science][Medline]
56. Yamauchi T, Kawai Y, Ueda T. Enhanced DNA excision repair in CCRF-CEM cells resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea, quantitated using the single cell gel electrophoresis (comet) assay. Biochem. Pharmacol. (2003) 66:939–946.[CrossRef][Web of Science][Medline]
57. Friedmann B, Caplin M, Hartley JA, Hochhauser D. Modulation of DNA repair in vitro after treatment with chemotherapeutic agents by the epidermal growth factor receptor inhibitor gefitinib (ZD1839). Clin. Cancer Res. (2004) 10:6476–6486.
58. Suggitt M, Fearnley J, Swaine DJ, Volpato M, Phillips RM, Bibby MC, Loadman PM, Anderson D. Comet assay and flow cytometry analysis of DNA repair in normal and cancer cells treated with known mutagens with different mechanisms of action. Teratog. Carcinog. Mutagen. (2003) (Suppl. 2):13–29.
59. Wozniak K, Kolacinska A, Blasinska-Morawiec M, Morawiec-Bajda A, Morawiec Z, Zadrozny M, Blasiak J. The DNA-damaging potential of tamoxifen in breast cancer and normal cells. Arch. Toxicol. (2007) 81:519–527.[CrossRef][Web of Science][Medline]
60. Liu X, Pisha E, Tonetti DA, Yao D, Li Y, Yao J, Burdette JE, Bolton JL. Antiestrogenic and DNA damaging effects induced by tamoxifen and toremifene metabolites. Chem. Res. Toxicol. (2003) 16:832–837.[Web of Science][Medline]
61. Zoli W, Ricotti L, Tesei A, Ulivi P, Gasperi Campani A, Fabbri F, Gunelli R, Frassineti GL, Amadori D. Schedule-dependent cytotoxic interaction between epidoxorubicin and gemcitabine in human bladder cancer cells in vitro. Clin. Cancer Res. (2004) 10:1500–1507.
62. Bruce IA, Slevin NJ, Homer JJ, McGown AT, Ward TH. Synergistic effects of imatinib (STI 571) in combination with chemotherapeutic drugs in head and neck cancer. Anticancer Drugs (2005) 16:719–726.[CrossRef][Medline]
63. Khaitan D, Chandna S, Arya MB, Dwarakanath BS. Differential mechanisms of radiosensitization by 2-deoxy-D-glucose in the monolayers and multicellular spheroids of a human glioma cell line. Cancer Biol. Ther. (2006) 5:1142–1151.[Web of Science][Medline]
64. Rousset N, Keminon E, Eleouet S, Le Neel T, Auget JL, Vonarx V, Carre J, Lajat Y, Patrice T. Use of alkaline comet assay to assess DNA repair after m-THPC-PDT. J. Photochem. Photobiol. B. (2000) 56:118–131.[CrossRef][Medline]
65. Guan H, Liu X, Peng W, Cao R, Ma Y, Chen H, Xu A. Beta-carboline derivatives: novel photosensitizers that intercalate into DNA to cause direct DNA damage in photodynamic therapy. Biochem. Biophys. Res. Commun. (2006) 342:894–901.[CrossRef][Web of Science][Medline]
66. Faust F, Kassie F, Knasmuller S, Boedecker RH, Mann M, Mersch-Sundermann V. The use of the alkaline comet assay with lymphocytes in human biomonitoring studies. Mutat. Res. (2004) 566:209–229.[CrossRef][Web of Science][Medline]
67. Møller P. The alkaline comet assay: towards validation in biomonitoring of DNA damaging exposures. Basic Clin. Pharmacol. Toxicol. (2006) 98:336–345.[CrossRef][Web of Science][Medline]
68. Palyvoda O, Polanska J, Wygoda A, Rzeszowska-Wolny J. DNA damage and repair in lymphocytes of normal individuals and cancer patients: studies by the comet assay and micronucleus tests. Acta Biochim. Pol. (2003) 50:181–190.[Web of Science][Medline]
69. Smith TR, Miller MS, Lohman KK, Case LD, Hu JJ. DNA damage and breast cancer risk. Carcinogenesis (2003) 24:883–889.
70. Rajeswari N, Ahuja YR, Malini U, Chandrashekar S, Balakrishna N, Rao KV, Khar A. Risk assessment in first degree female relatives of breast cancer patients using the alkaline comet assay. Carcinogenesis (2000) 21:557–561.
71. Sanchez C, Clementi M, Benitez D, Contreras H, Huidobro C, Castellon E. Effect of GnRH analogs on the expression of TrkA and p75 neurotrophin receptors in primary cell cultures from human prostate adenocarcinoma. Prostate (2005) 65:195–202.[CrossRef][Web of Science][Medline]
72. Lin X, Wood CG, Shao L, Huang M, Yang H, Dinney CP, Wu X. Risk assessment of renal cell carcinoma using alkaline comet assay. Cancer (2007) 110:282–288.[Medline]
73. Schabath MB, Grossman HB, Delclos GL, Hernandez LM, Day RS, Davis BR, Lerner SP, Spitz MR, Wu X. Dietary carotenoids and genetic instability modify bladder cancer risk. J. Nutr. (2004) 134:3362–3369.
74. Lou J, He J, Zheng W, Jin L, Chen Z, Chen S, Lin Y, Xu S. Investigating the genetic instability in the peripheral lymphocytes of 36 untreated lung cancer patients with comet assay and micronucleus assay. Mutat. Res. (2007) 617:104–110.[Web of Science][Medline]
75. Nascimento PA, da Silva MA, Oliveira EM, Suzuki MF, Okazaki K. Evaluation of radioinduced damage and repair capacity in blood lymphocytes of breast cancer patients. Braz. J. Med. Biol. Res. (2001) 34:165–176.[Web of Science][Medline]
76. Alapetite C, Thirion P, de la Rochefordiere A, Cosset JM, Moustacchi E. Analysis by alkaline comet assay of cancer patients with severe reactions to radiotherapy: defective rejoining of radioinduced DNA strand breaks in lymphocytes of breast cancer patients. Int. J. Cancer (1999) 83:83–90.[Web of Science][Medline]
77. Blasiak J, Arabski M, Krupa R, Wozniak K, Rykala J, Kolacinska A, Morawiec Z, Drzewoski J, Zadrozny M. Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer. Mutat. Res. (2004) 554:139–148.[Web of Science][Medline]
78. Rajaee-Behbahani N, Schmezer P, Risch A, et al. Altered DNA repair capacity and bleomycin sensitivity as risk markers for non-small cell lung cancer. Int. J. Cancer (2001) 95:86–91.[CrossRef][Web of Science][Medline]
79. Wei Z, Lifen J, Jiliang H, Jianlin L, Baohong W, Hongping D. Detecting DNA repair capacity of peripheral lymphocytes from cancer patients with UVC challenge test and bleomycin challenge test. Mutagenesis (2005) 20:271–277.
80. Jianlin L, Jiliang H, Lifen J, Wei Z, Baohong W, Hongping D. Measuring the genetic damage in cancer patients during radiotherapy with three genetic end-points. Mutagenesis (2004) 19:457–464.
81. Kopjar N, Garaj-Vrhovac V, Milas I. Assessment of chemotherapy-induced DNA damage in peripheral blood leukocytes of cancer patients using the alkaline comet assay. Teratog. Carcinog. Mutagen. (2002) 22:13–30.[CrossRef][Web of Science][Medline]
82. Almeida GM, Duarte TL, Steward WP, Jones GD. Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay. DNA Repair (2006) 5:219–225.[CrossRef][Medline]
83. Plummer ER, Middleton MR, Jones C, et al. Temozolomide pharmacodynamics in patients with metastatic melanoma: DNA damage and activity of repair enzymes O6-alkylguanine alkyltransferase and poly(ADP-ribose) polymerase-1. Clin. Cancer Res. (2005) 11:3402–3409.
84. Oppitz U, Schulte S, Stopper H, Baier K, Muller M, Wulf J, Schakowski R, Flentje M. In vitro radiosensitivity measured in lymphocytes and fibroblasts by colony formation and comet assay: comparison with clinical acute reactions to radiotherapy in breast cancer patients. Int. J. Radiat. Biol. (2002) 78:611–616.[CrossRef][Web of Science][Medline]
85. Nadin SB, Vargas-Roig LM, Drago G, Ibarra J, Ciocca DR. DNA damage and repair in peripheral blood lymphocytes from healthy individuals and cancer patients: a pilot study on the implications in the clinical response to chemotherapy. Cancer Lett. (2006) 239:84–97.[CrossRef][Web of Science][Medline]
86. Cao J, Liu Y, Sun H, Cheng G, Pang X, Zhou Z. Chromosomal aberrations, DNA strand breaks and gene mutations in nasopharyngeal cancer patients undergoing radiation therapy. Mutat. Res. (2002) 504:85–90.[Web of Science][Medline]
87. Braybrooke JP, Houlbrook S, Crawley JE, Propper DJ, O'Byrne KJ, Stratford IJ, Harris AL, Shuker DE, Talbot DC. Evaluation of the alkaline comet assay and urinary 3-methyladenine excretion for monitoring DNA damage in melanoma patients treated with dacarbazine and tamoxifen. Cancer Chemother. Pharmacol. (2000) 45:111–119.[CrossRef][Web of Science][Medline]
88. Hartley JM, Spanswick VJ, Gander M, Giacomini G, Whelan J, Souhami RL, Hartley JA. Measurement of DNA cross-linking in patients on ifosfamide therapy using the single cell gel electrophoresis (comet) assay. Clin. Cancer Res. (1999) 5:507–512.
89. Reed E, Yuspa SH, Zwelling LA, Ozols RF, Poirier MC. Quantitation of cis-diamminedichloroplatinum II (cisplatin)-DNA-intrastrand adducts in testicular and ovarian cancer patients receiving cisplatin chemotherapy. J. Clin. Invest. (1986) 77:545–550.[Web of Science][Medline]
90. Parker RJ, Gill I, Tarone R, Vionnet JA, Grunberg S, Muggia FM, Reed E. Platinum-DNA damage in leukocyte DNA of patients receiving carboplatin and cisplatin chemotherapy, measured by atomic absorption spectrometry. Carcinogenesis (1991) 12:1253–1258.
91. Bosken CH, Wei Q, Amos CI, Spitz MR. An analysis of DNA repair as a determinant of survival in patients with non-small-cell lung cancer. J. Natl. Cancer Inst. (2002) 94:1091–1099.
92. Angerer J, Ewers U, Wilhelm M. Human biomonitoring: state of the art. Int. J. Hyg. Environ. Health (2007) 210:201–228.[CrossRef][Web of Science][Medline]
93. Isa AY, Ward TH, West CM, Slevin NJ, Homer JJ. Hypoxia in head and neck cancer. Br. J. Radiol. (2006) 79:791–798.
94. Tatum JL, Kelloff GJ, Gillies RJ, et al. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int. J. Radiat. Biol. (2006) 82:699–757.[CrossRef][Web of Science][Medline]
95. Milosevic M, Fyles A, Hedley D, Hill R. The human tumor microenvironment: invasive (needle) measurement of oxygen and interstitial fluid pressure. Semin. Radiat. Oncol. (2004) 14:249–258.[CrossRef][Web of Science][Medline]
96. Taube SE, Jacobson JW, Lively TG. Cancer diagnostics: decision criteria for marker utilization in the clinic. Am. J. Pharmacogenomics (2005) 5:357–364.[CrossRef][Web of Science][Medline]
97. McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM. Statistics subcommittee of NCI-EORTC working group on cancer diagnostics. Reporting recommendations for tumor marker prognostic studies (REMARK). Breast Cancer Res. Treat. (2006) 100:229–235.[CrossRef][Web of Science][Medline]
98. Hagmar L, Bonassi S, Strömberg U, Brøgger A, Knudsen LE, Norppa H, Reuterwall C. the European Study Group on Cytogenetic Biomarkers and Health. Chromosome aberrations in lymphocytes predict human cancer: a report from the European study group on cytogenetic biomarkers and health (ESCH). Cancer Res. (1998) 58:4117–4121.
99. ESCODD (European Standards Committee on Oxidative DNA Damage). Gedik CM, Collins A. Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study. FASEB J. (2005) 19:82–84.
Received on October 30, 2007; revised on November 30, 2007; accepted on November 30, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Zainol, J. Stoute, G. M. Almeida, A. Rapp, K. J. Bowman, G. D. D. Jones, and ECVAG Introducing a true internal standard for the Comet assay to minimize intra- and inter-experiment variability in measures of DNA damage and repair Nucleic Acids Res., October 14, 2009; (2009) gkp826v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Milkovic, V. Garaj-Vrhovac, M. Ranogajec-Komor, S. Miljanic, G. Gajski, Z. Knezevic, and N. Beck Primary DNA Damage Assessed With the Comet Assay and Comparison to the Absorbed Dose of Diagnostic X-rays in Children International Journal of Toxicology, September 1, 2009; 28(5): 405 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Herrera, G. Dominguez, J. M. Garcia, C. Pena, C. Jimenez, J. Silva, V. Garcia, I. Gomez, R. Diaz, P. Martin, et al. Differences in Repair of DNA Cross-links between Lymphocytes and Epithelial Tumor Cells from Colon Cancer Patients Measured In vitro with the Comet Assay Clin. Cancer Res., September 1, 2009; 15(17): 5466 - 5472. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||