- Email: [email protected]
Measurement of incorporation of bromodeoxyuridine into DNA by high performance liquid chromatography using a novel fluorescent labelling technique
hr. J. Radiarion Oncology Bid. Phys., Vol. 22. pp. 485-%87 Printed 1” the U.S A. All nghts reserved.
Copywght
0360-3016/92 $5.00 + .oO Q 1992 Pergamon Press plc
??Session B: Biochemical Modification of Therapeutic Response
MEASUREMENT OF INCORPORATION OF BROMODEOXYURIDINE INTO DNA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING A NOVEL FLUORESCENT LABELLING TECHNIQUE MICHAEL R. L. STRATFORD, PH.D. AND MADELEINEF. DENNIS, M.A. The Gray Laboratory of the Cancer Research Campaign, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, U.K. 5’.Bromo-2-deoxyuridine (BUdR) is a halogenated pyrimldine analogue that is an efficient radiosensitizer through its incorporation into DNA in place of thymidine. Radiosensitization is proportional to percentage replacement and we present here a novel derivatization technique that specifically labels the thymldine and BUdR with 4-bromomethyl-7-methoxycoumarin (BrMMC) to give the highly fluorescent coumarin derivatives which are quantitated using high performance liquid chromatography (HPLC). This allows for a simple single-stage DNA hydrolysis and sensitive peak detection. Data are presented showing the incorporation with time of BUdR into the DNA of Chinese hamster V79 cells. Attention is also drawn to the care needed in the selection of enzymes required for DNA digestion. Bromodeoxyuridine, BUdR, DNA, Incorporation, HPLC, Fluorescent derivative, 4-bromomethyl-7.methoxycoumarin. after adding BUdR, aliquots (4 x lo6 cells) were taken, spun, and washed with PBS. DNA was digested using a modification of the technique of Belanger et al. (1). The RNA digestion was omitted and 470 K units of DNase I and 0.027 units of phosphodiesterase 1 type IV* were used with a 1 hr incubation in a total volume of 1 ml of 50 mM potassium phosphate buffer, pH 7.6, containing 5 mM magnesium sulphate. The sample was then centrifuged and a 200 ~1 aliquot applied to a Cl8 cartridge (100 mg size)? previously equilibrated with 2 ml acetonitrile, followed by 1 ml water and 1 ml of incubation buffer. After washing with 200 pl water, the nucleosides were eluted with 0.6 ml 60% acetonitrile, then taken to dryness under nitrogen. To form the fluorescent coumarin derivative, 50 ~1 dimethylsulfoxide (DMSO) was added to each tube, followed by 2 mg powdered K,CO,, 20 ~1 of the crown ether l&crown6* and 200 ~1 BrMMC$ (recrystallised from acetonitrile) (both 2.5 mM in acetonitrile), with thorough mixing after each addition. The acetonitrile was then evaporated under nitrogen and 200 pl 75% acetonitrilelwater added. The samples were then centrifuged, 190 ~1 of the supernatant was added to 10 p,l 0.5 M acetic acid, and analyzed by HPLC, using a Cl8 cartridges (5 mm x 10 cm), and detection was by fluorimetry** with excitation at 320 nm and
INTRODUCTION The effect of halogenated pyrimidines such as 5’-iodo- and 5’-bromo-2-deoxyuridine on radiation sensitivity, following incorporation into DNA, was first described in 1959 (1 I), but there is continuing interest in their clinical use (3, 8). Radiosensitivity is dependent on the extent of incorporation (5), but effects have been seen with levels of replacement of thymidine of less than 2% (2). A number of techniques have been used to determine the degree of replacement, including neutron activation analysis (6), radiolabelling (6, 7), and chromatographic measurement of BUdR and thymidine after enzymatic digestion of the DNA (4, IO). We describe here the use of the reaction of 4-bromomethyl-7-methoxycoumarin (BrMMC) with the imide nitrogen in thymidine and BUdR to give fluorescent products (11). This allows a simplified single step enzymatic degradation of the DNA and provides sensitive detection of BUdR. METHODS AND MATERIALS Cells (V79 379A Chinese hamster fibroblasts) were incubated at 5 X lO’/ml in Eagles minimum essential medium + 7.5% fetal calf serum at 37°C. At various times
tSep-Pak Vat, Millipore (UK) Ltd., Watford, U.K.
Presented at the 7th International Conference on Chemical Modifiers of Cancer Treatment, 2-5 February 1991.
*Sigma Chemical Co., Poole, U.K. $.FSA, Loughborough, U.K. §Novapak, Millipore (UK) Ltd. Watford, U.K. **LSI, Perkin Elmer, Beaconsfield, U.K.
Reprint requests to: M.R.L. Stratford, Ph.D. Supported by the Cancer Research Campaign. Accepted for publication 3 July 1991. 485
I. J. Radiation Oncology 0 Biology 0 Physics
Volume 22, Number 3, 1992
Fig. 2. Incorporation of BUdR (8 FM) into Chinese hamster fibroblast DNA with time.
12
8
4 time
(min)
Fig. 1. (a) HPLC chromatograms
of the MMC derivatives of digests of cells exposed to 8 pM BUdR for 8 hr. (b) As above in absence of BUdR.
emission monitored at 394 nm. The eluents were water (A) and 75% acetonitrile and water (B), pumped at 2 ml/min, using linear gradients from 28-32% B, O-4 min, hold at 32% for 4 min, and 32-80% B, 8-9 min.
RESULTS AND DISCUSSION Figure 1 shows chromatograms obtained following extraction and derivatization of cells incubated (a) with or (b) without 8 ~_LMBUdR and illustrates the specificity of the reaction for thymidine and BUdR. All other peaks are reagent-related. Digests of cells incubated with BUdR for various times were derivatized, and the relative amounts of BUdR and thymidine calculated by comparison with BUdR and thymidine standard areas. The percentage of thymidine replacement was calculated, and the results are presented in Figure 2. These agree with published data, for example those of Ling and Ward (7). We tried a number of other digestion procedures before using the short incubation with just DNase and phosphodiesterase described here. As noted by Belanger (l), sufficient phosphatase activity is present in the enzymes to completely degrade nucleotides to nucleosides. However, t?Bond-Elut,
Jones Chromatography,
Hengoed,
Wales.
we found the 10 hr incubation used by these authors led to additional peaks being formed that were apparent using absorbance detection, which we used for our initial work, Specific addition of alkaline phosphatase also resulted in the creation of additional peaks, perhaps due to deaminase activity as noted by Singhal and Landes (9). It was important to remove the buffer from the digest prior to derivatization, or the yield was greatly reduced; this was the main function of the Seppak stage. Initially, we tried an altemative Cl8 extraction columntt , but this was not sufficiently retentive for the rather polar nucleosides. It was also found that reuse of the columns sometimes gave very low derivatization yield, despite extensive washing and conditioning, although the reason for this was not clear. The DMSO served to ensure the nucleosides were fully dissolved in the derivatization solvent; its omission led to lower yields of derivative from cell digests compared with standards. Contrary to the data of Yoshida et al. (12), we did not find the yield of fluorescent product to be increased by refluxing; however, evaporating the acetonitrile off and redissolving in 75% acetonitrile had a dual benefit. The drying down appeared to increase slightly the derivatization efficiency, while the partly aqueous solvent served to remove some of the reagent that otherwise gave rise occasionally to blockage of the analytical column after injection into the predominantly aqueous eluent. The acidification with acetic acid increases the stability of the derivative; however, it cannot be added directly to the whole sample as this results in the appearance of additional interfering peaks. A number of workers have published data on incorporation of halogenated pyrimidines into DNA using differing techniques. Although for 5’-iodo-deoxyuridine, levels as low as 0.2% have been reported using U.V. detection (l), because BUdR is more hydrophillic, it is more difficult to separate from endogenous material, and published values do not go below 5% (4). It is possible to use radiolabelled BUdR (7), but unless these are combined with
High performance liquid chromatography 0 M. R. L.
chromatographic separation, then dehalogenation can lead to errors (1). Finally, using GC-MS, it is possible to measure low levels of incorporation (lo), but this technique is not widely available. Using the method described here, it is possible to carry out a single one-stage digestion of the DNA because of the
STRATFORD
ANDM. F. DENNIS
487
specificity of the derivatization reaction. It is straightforward to measure less than 1% incorporation from 4 X lo6 cells, using only 20% of the digest. Because of the high intrinsic sensitivity of fluorescence detection, the limitation on detection is governed primarily by reagent related interferences which may be amenable to further reduction.
REFERENCES 1. Belanger, K.; Collins, J. M.; Klecker, R. W. J. Technique for detection of DNA nucleobases by reversed phase high performance liquid chromatography optimised for quantitative determination of thymidine substitution by iododeoxyuridine. J. Chromatogr. 4175763; 1987. 2. Berry, R. J.; Andrews, J. R. Modification of the radiation effect on the reproductive capacity of tumor cells in vivo with pharmacological agents. Radiat. Res. 16:84-88; 1962. 3. Hegarty, T. J.; Thornton, A. F.; Diaz, R. F.; Chandler, W. F.; Ensminger, W. D.; Junck, L.; Page, M. A.; Gebarski, S. S.; Hood, T. W.; Stetson, P. L.; Tankanow, R. M.; McKeever, P. E.; Lichter, A. S.; Greenberg, H. S. Intra-arterial bromodeoxyuridine and radiosensitization of malignant gliomas. Int. J. Radiat. Oncol. Biol. Phys. 19:421428; 1990. 4. Iliakis, G.; Kurtzman, S.; Pantelias, G.; Okayasu, R. Mechanism of radiosensitization by halogenated pyrimidine: effect of BrdU on radiation induction of DNA and chromosome damage and its correlation with cell killing. Radiat. Res. 119:286-304; 1989. 5. Kinsella, T. J.; Mitchell, J. B.; Russo, A.; Morstyn, C.; Glatstein, E. The use of halogenated thymidine analogues as clinical radiosensitizers. Int. J. Radiat. Oncol. Biol. Phys. 10: 1399-1406; 1984. 6. Laster, B. H.; Popenoe, E. A.; Wielopolski, L.; Commerford, S. L.; Gahbauer, R.; Goodman, J.; Meek, A.; Fairchild, R. G. Analysis of 5-iodo-2-deoxyuridine incorporation
7.
8.
9.
10.
11. 12.
in murine melanoma for photon activation therapy. Radiother. Oncol. 19:169-178; 1990. Ling, L. L.; Ward, J. F. Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution. Radiat. Res. 121:76-83; 1990. Mitchell, J. B.; Russo, A.; Cook, J. A.; Strauss, K. L.; Glatstein, E. Radiobiology and clinical application of halogenated pyrimidine radiosensitizers. Int. J. Radiat. Biol. 56: 827-836; 1989. Singhal, R. P.; Landes, J. P. High performance liquid chromatographic analysis of DNA composition and DNA modification by chloroacetaldehyde. J. Chromatogr. 458: 117128; 1988. Stetson, P. L.; Maybaum, J.; Shukla, U. S.; Ensminger, W. D. Simultaneous determination of thymine and 5-bromouracil in DNA hydrolysates using gas chromatography-mass spectrometry with selected-ion-monitoring. J. Chromatogr. 375:1-9; 1986. Szybalski, W.; Djordjevic, B. Radiation sensitivity of chemically modified human cells. Genetics 44:540-541; 1959. Yoshida, S.; Hirose, S.; Iwamoto, M. Use of 4-bromomethyl-7-methoxycoumarin for derivatization of pyrimidine compounds in serum analysed by high performance liquid chromatography with fluorometric detection. J. Chromatogr. 383:61-68; 1986.
Copywght
0360-3016/92 $5.00 + .oO Q 1992 Pergamon Press plc
??Session B: Biochemical Modification of Therapeutic Response
MEASUREMENT OF INCORPORATION OF BROMODEOXYURIDINE INTO DNA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING A NOVEL FLUORESCENT LABELLING TECHNIQUE MICHAEL R. L. STRATFORD, PH.D. AND MADELEINEF. DENNIS, M.A. The Gray Laboratory of the Cancer Research Campaign, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, U.K. 5’.Bromo-2-deoxyuridine (BUdR) is a halogenated pyrimldine analogue that is an efficient radiosensitizer through its incorporation into DNA in place of thymidine. Radiosensitization is proportional to percentage replacement and we present here a novel derivatization technique that specifically labels the thymldine and BUdR with 4-bromomethyl-7-methoxycoumarin (BrMMC) to give the highly fluorescent coumarin derivatives which are quantitated using high performance liquid chromatography (HPLC). This allows for a simple single-stage DNA hydrolysis and sensitive peak detection. Data are presented showing the incorporation with time of BUdR into the DNA of Chinese hamster V79 cells. Attention is also drawn to the care needed in the selection of enzymes required for DNA digestion. Bromodeoxyuridine, BUdR, DNA, Incorporation, HPLC, Fluorescent derivative, 4-bromomethyl-7.methoxycoumarin. after adding BUdR, aliquots (4 x lo6 cells) were taken, spun, and washed with PBS. DNA was digested using a modification of the technique of Belanger et al. (1). The RNA digestion was omitted and 470 K units of DNase I and 0.027 units of phosphodiesterase 1 type IV* were used with a 1 hr incubation in a total volume of 1 ml of 50 mM potassium phosphate buffer, pH 7.6, containing 5 mM magnesium sulphate. The sample was then centrifuged and a 200 ~1 aliquot applied to a Cl8 cartridge (100 mg size)? previously equilibrated with 2 ml acetonitrile, followed by 1 ml water and 1 ml of incubation buffer. After washing with 200 pl water, the nucleosides were eluted with 0.6 ml 60% acetonitrile, then taken to dryness under nitrogen. To form the fluorescent coumarin derivative, 50 ~1 dimethylsulfoxide (DMSO) was added to each tube, followed by 2 mg powdered K,CO,, 20 ~1 of the crown ether l&crown6* and 200 ~1 BrMMC$ (recrystallised from acetonitrile) (both 2.5 mM in acetonitrile), with thorough mixing after each addition. The acetonitrile was then evaporated under nitrogen and 200 pl 75% acetonitrilelwater added. The samples were then centrifuged, 190 ~1 of the supernatant was added to 10 p,l 0.5 M acetic acid, and analyzed by HPLC, using a Cl8 cartridges (5 mm x 10 cm), and detection was by fluorimetry** with excitation at 320 nm and
INTRODUCTION The effect of halogenated pyrimidines such as 5’-iodo- and 5’-bromo-2-deoxyuridine on radiation sensitivity, following incorporation into DNA, was first described in 1959 (1 I), but there is continuing interest in their clinical use (3, 8). Radiosensitivity is dependent on the extent of incorporation (5), but effects have been seen with levels of replacement of thymidine of less than 2% (2). A number of techniques have been used to determine the degree of replacement, including neutron activation analysis (6), radiolabelling (6, 7), and chromatographic measurement of BUdR and thymidine after enzymatic digestion of the DNA (4, IO). We describe here the use of the reaction of 4-bromomethyl-7-methoxycoumarin (BrMMC) with the imide nitrogen in thymidine and BUdR to give fluorescent products (11). This allows a simplified single step enzymatic degradation of the DNA and provides sensitive detection of BUdR. METHODS AND MATERIALS Cells (V79 379A Chinese hamster fibroblasts) were incubated at 5 X lO’/ml in Eagles minimum essential medium + 7.5% fetal calf serum at 37°C. At various times
tSep-Pak Vat, Millipore (UK) Ltd., Watford, U.K.
Presented at the 7th International Conference on Chemical Modifiers of Cancer Treatment, 2-5 February 1991.
*Sigma Chemical Co., Poole, U.K. $.FSA, Loughborough, U.K. §Novapak, Millipore (UK) Ltd. Watford, U.K. **LSI, Perkin Elmer, Beaconsfield, U.K.
Reprint requests to: M.R.L. Stratford, Ph.D. Supported by the Cancer Research Campaign. Accepted for publication 3 July 1991. 485
I. J. Radiation Oncology 0 Biology 0 Physics
Volume 22, Number 3, 1992
Fig. 2. Incorporation of BUdR (8 FM) into Chinese hamster fibroblast DNA with time.
12
8
4 time
(min)
Fig. 1. (a) HPLC chromatograms
of the MMC derivatives of digests of cells exposed to 8 pM BUdR for 8 hr. (b) As above in absence of BUdR.
emission monitored at 394 nm. The eluents were water (A) and 75% acetonitrile and water (B), pumped at 2 ml/min, using linear gradients from 28-32% B, O-4 min, hold at 32% for 4 min, and 32-80% B, 8-9 min.
RESULTS AND DISCUSSION Figure 1 shows chromatograms obtained following extraction and derivatization of cells incubated (a) with or (b) without 8 ~_LMBUdR and illustrates the specificity of the reaction for thymidine and BUdR. All other peaks are reagent-related. Digests of cells incubated with BUdR for various times were derivatized, and the relative amounts of BUdR and thymidine calculated by comparison with BUdR and thymidine standard areas. The percentage of thymidine replacement was calculated, and the results are presented in Figure 2. These agree with published data, for example those of Ling and Ward (7). We tried a number of other digestion procedures before using the short incubation with just DNase and phosphodiesterase described here. As noted by Belanger (l), sufficient phosphatase activity is present in the enzymes to completely degrade nucleotides to nucleosides. However, t?Bond-Elut,
Jones Chromatography,
Hengoed,
Wales.
we found the 10 hr incubation used by these authors led to additional peaks being formed that were apparent using absorbance detection, which we used for our initial work, Specific addition of alkaline phosphatase also resulted in the creation of additional peaks, perhaps due to deaminase activity as noted by Singhal and Landes (9). It was important to remove the buffer from the digest prior to derivatization, or the yield was greatly reduced; this was the main function of the Seppak stage. Initially, we tried an altemative Cl8 extraction columntt , but this was not sufficiently retentive for the rather polar nucleosides. It was also found that reuse of the columns sometimes gave very low derivatization yield, despite extensive washing and conditioning, although the reason for this was not clear. The DMSO served to ensure the nucleosides were fully dissolved in the derivatization solvent; its omission led to lower yields of derivative from cell digests compared with standards. Contrary to the data of Yoshida et al. (12), we did not find the yield of fluorescent product to be increased by refluxing; however, evaporating the acetonitrile off and redissolving in 75% acetonitrile had a dual benefit. The drying down appeared to increase slightly the derivatization efficiency, while the partly aqueous solvent served to remove some of the reagent that otherwise gave rise occasionally to blockage of the analytical column after injection into the predominantly aqueous eluent. The acidification with acetic acid increases the stability of the derivative; however, it cannot be added directly to the whole sample as this results in the appearance of additional interfering peaks. A number of workers have published data on incorporation of halogenated pyrimidines into DNA using differing techniques. Although for 5’-iodo-deoxyuridine, levels as low as 0.2% have been reported using U.V. detection (l), because BUdR is more hydrophillic, it is more difficult to separate from endogenous material, and published values do not go below 5% (4). It is possible to use radiolabelled BUdR (7), but unless these are combined with
High performance liquid chromatography 0 M. R. L.
chromatographic separation, then dehalogenation can lead to errors (1). Finally, using GC-MS, it is possible to measure low levels of incorporation (lo), but this technique is not widely available. Using the method described here, it is possible to carry out a single one-stage digestion of the DNA because of the
STRATFORD
ANDM. F. DENNIS
487
specificity of the derivatization reaction. It is straightforward to measure less than 1% incorporation from 4 X lo6 cells, using only 20% of the digest. Because of the high intrinsic sensitivity of fluorescence detection, the limitation on detection is governed primarily by reagent related interferences which may be amenable to further reduction.
REFERENCES 1. Belanger, K.; Collins, J. M.; Klecker, R. W. J. Technique for detection of DNA nucleobases by reversed phase high performance liquid chromatography optimised for quantitative determination of thymidine substitution by iododeoxyuridine. J. Chromatogr. 4175763; 1987. 2. Berry, R. J.; Andrews, J. R. Modification of the radiation effect on the reproductive capacity of tumor cells in vivo with pharmacological agents. Radiat. Res. 16:84-88; 1962. 3. Hegarty, T. J.; Thornton, A. F.; Diaz, R. F.; Chandler, W. F.; Ensminger, W. D.; Junck, L.; Page, M. A.; Gebarski, S. S.; Hood, T. W.; Stetson, P. L.; Tankanow, R. M.; McKeever, P. E.; Lichter, A. S.; Greenberg, H. S. Intra-arterial bromodeoxyuridine and radiosensitization of malignant gliomas. Int. J. Radiat. Oncol. Biol. Phys. 19:421428; 1990. 4. Iliakis, G.; Kurtzman, S.; Pantelias, G.; Okayasu, R. Mechanism of radiosensitization by halogenated pyrimidine: effect of BrdU on radiation induction of DNA and chromosome damage and its correlation with cell killing. Radiat. Res. 119:286-304; 1989. 5. Kinsella, T. J.; Mitchell, J. B.; Russo, A.; Morstyn, C.; Glatstein, E. The use of halogenated thymidine analogues as clinical radiosensitizers. Int. J. Radiat. Oncol. Biol. Phys. 10: 1399-1406; 1984. 6. Laster, B. H.; Popenoe, E. A.; Wielopolski, L.; Commerford, S. L.; Gahbauer, R.; Goodman, J.; Meek, A.; Fairchild, R. G. Analysis of 5-iodo-2-deoxyuridine incorporation
7.
8.
9.
10.
11. 12.
in murine melanoma for photon activation therapy. Radiother. Oncol. 19:169-178; 1990. Ling, L. L.; Ward, J. F. Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution. Radiat. Res. 121:76-83; 1990. Mitchell, J. B.; Russo, A.; Cook, J. A.; Strauss, K. L.; Glatstein, E. Radiobiology and clinical application of halogenated pyrimidine radiosensitizers. Int. J. Radiat. Biol. 56: 827-836; 1989. Singhal, R. P.; Landes, J. P. High performance liquid chromatographic analysis of DNA composition and DNA modification by chloroacetaldehyde. J. Chromatogr. 458: 117128; 1988. Stetson, P. L.; Maybaum, J.; Shukla, U. S.; Ensminger, W. D. Simultaneous determination of thymine and 5-bromouracil in DNA hydrolysates using gas chromatography-mass spectrometry with selected-ion-monitoring. J. Chromatogr. 375:1-9; 1986. Szybalski, W.; Djordjevic, B. Radiation sensitivity of chemically modified human cells. Genetics 44:540-541; 1959. Yoshida, S.; Hirose, S.; Iwamoto, M. Use of 4-bromomethyl-7-methoxycoumarin for derivatization of pyrimidine compounds in serum analysed by high performance liquid chromatography with fluorometric detection. J. Chromatogr. 383:61-68; 1986.