185
Mutation Research, DNA Repair, 254 (1991) 185-190 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100060Y MUTDNA 06423
Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom-syndrome fibroblasts Yves Pommier, Thomas M. Ranger, Donna Kerrigan and Kenneth H. Kraemer Laboratory of Molecular Pharmacology (YP, DK) and Laboratory of Molecular Carcinogenesis (TMR, KK), National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) (Received 1 March 1990) (Revision received 3 August 1990) (Accepted 10 August 1990)
Keywords: Bloom-syndrome fibroblasts; DNA strand breakage; Bromodeoxyuridine; Topoisomerase II
Smmnary Cells from patients with Bloom syndrome, a cancer-prone disorder with cutaneous photosensitivity and spontaneous chromosome breakage, exhibit an abnormally increased number of sister-chromatid exchanges following treatment with 5-bromodeoxyuridine (BrdU). This effect has been postulated to be mediated by abnormal topoisomerase II activity. We used alkaline elution to measure DNA single-strand breakage following prolonged exposure to BrdU. Five-day exposure to BrdU produced equal numbers of alkali-labile sites in normal and Bloom-syndrome fibroblasts. These breaks were not protein-associated but were produced by alkali. Treatment with topoisomerase II inhibitors induced similar frequencies of DNA single-strand breaks in normal and Bloom-syndrome fibroblasts. These findings imply that BrdU incorporation into cellular DNA induces alkali-labile DNA lesions that are independent of topoisomerase II activity in Bloom and normal cells.
Bloom syndrome is a rare autosomal recessive disease with a high cancer risk, sun sensitivity, immunodeficiency and growth retardation (German, 1969). Cells from patients with Bloom-syndrome are characterized by an elevated level of spontaneous chromosome breakage and sisterchromatid exchanges (SCE). The chromosome breakage and SCE increase upon exposure to 5bromodeoxyuridine (BrdU) (Chaganti et al., 1974; Heartlein et al., 1987; Tsuji et al., 1988). BrdU has
Correspondence: Dr. Yves Pommier, Bldg. 37, Rm. 5C27, National Cancer Institute, Bethesda, MD 20892 (U.S.A.), Telephone: (301)496-5944.
also been reported to produce alkaline-labile DNA strand breaks (Dillehay et al., 1984; Fornace et al., 1986). Because it has been reported that the induction of SCE by antitumor topoisomerase II inhibitors may be related to the trapping of topoisomerase II-mediated DNA breaks (Pommier et al., 1985), it has been suggested that the induction of SCE by BrdU may result from abnormal topoisomerase II activity in Bloom-syndrome cells (Heartlein et al., 1987). Other reports suggested a DNA ligase I deficiency in Bloom-syndrome cells (Willis et al., 1987; Chan et al., 1987; Ranger and Kraemer, 1989) and abnormal antigenicity of uracil DNA glycosylase (Volberg et al., 1987). We examined the role of DNA topoisomerase
186
II in BrdU-induced DNA breakage in Bloom-syndrome and normal fibroblasts. DNA topoisomerase II binds to DNA, produces a double-strand break while covalently binding to DNA termini and then rejoins the ends of the DNA following a topological unwinding (Wang, 1987; Pommier and Kohn, 1988; Liu, 1989). Topoisomerase II-induced DNA breaks are characteristically present under deproteinizing conditions and are not detectable under non-deproteinizing conditions (Pommier and Kohn, 1988; Covey et al., 1989). Antitumor topoisomerase II inhibitors, such as amsacrine (m-AMSA) and teniposide (VP-16) function by trapping the topoisomerase II-DNA complex after the DNA has been cut by the topoisomerase II but prior to the rejoining of the cut ends (Wang, 1987; Pommier and Kohn, 1988; Liu, 1989). The frequency of protein-linked DNA breaks following treatment with topoisomerase II inhibitors thus is a measure of the extent of topoisomerase II activity within the cells. In the present study we used alkaline elution to measure the induction of protein-linked and nonprotein-linked DNA breaks by BrdU in Bloomsyndrome and normal cells. We also assessed the topoisomerase II activity in these cells following treatment with topoisomerase II inhibitors.
Mutant Cell Repository, Coriel Institute for Medical Research, Camden, NJ. Cells were grown in monolayer cultures in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and 20 mM glutamine in a humidified atmosphere with 8% CO 2. Fibroblast doubling times were approx. 30 h (normal) and 40 h (Bloom syndrome). Suspension cultures of L1210 leukemia cells were maintained as previously described (Kohn et al., 1981). For alkaline elution experiments, normal and Bloom-syndrome fibroblast cell lines were plated at 1 × 105 cells/25 cm2 in medium containing 0.02 /~Ci/ml [14C]thymidine and grown for 2-5 days. L1210 cells used for internal standards were labeled for 16-24 h with 0.2 /~Ci/ml [3H]thymidine and chased in unlabeled medium for at least 2 h prior to use. In the BrdU experiments cells were grown in the presence of both BrdU and 10/~M FdU for 5 days (Heartlein et al., 1987) and maintained in the dark throughout the experiments. Topoisomerase inhibitors (m-AMSA, VP-16) were added to cell cultures such that the concentration of DMSO never exceeded 0.25%. Drug exposures were for 30 rain at 37°C, and drug treatments were terminated by rinsing cells twice with ice-cold Hanks' Balanced Salt Solution (HBSS) and then scraping them into the same buffer.
Materials and methods
Chemicals 5-Bromodeoxyuridine (BrdU) and fluorodeoxyuridine (FdU) were obtained from Sigma Chemical Co., St. Louis, MO. [Me-3H]Thymidine and [2-14C]thymidine (spec. act. 20 and 0.05 /~Ci/mmole, respectively), were purchased from NEN Research Products (Boston, MA). 4'-(9Acridinylamino)methanesulfon-m-anisidide (mAMSA) (NSC 249992) was obtained from the Pharmaceutical Resources Branch, Division of Cancer Treatment, NCI, Bethesda, MD. Etoposide (VP-16) was a gift from the Bristol Myers Co., Wallingford, MA. Stock solutions were prepared in dimethyl sulfoxide (DMSO) at 10 mM. Cell cultures, and BrdU treatment Simian virus 40 (SV40) transformed fibroblasts (normal: GM 00637; and Bloom syndrome: GM 08505A) were obtained from the Human Genetic
Determination of DNA single-strand break frequency DNA single-strand breaks were analyzed using alkaline elution carried out under deproteinizing conditions as described previously (Kohn et al., 1981). Briefly, aliquots of [14C]-labeled cells were mixed in ice-cold phosphate-buffered saline (PBS) with [3H]-labeled internal standard cells that had been irradiated with 300 rad ~,-radiation immediately before. Cells were then layered onto polycarbonate filters (2.0 /~m pore diameter, Nucleopore Corp., Pleasanton, CA) and were lysed with a solution containing 2% SDS, 0.1 M glycine, 0.025 M Na2EDTA, and 0.5 mg/ml proteinase K, pH 10 (SDS-Lysis). The lysis solution was washed with 5 ml of 0.02 M EDTA, pH 10, and the DNA was eluted with tetrapropylammonium hydroxide-EDTA, 0.1% SDS, pH 12.1 (or 12.6; Fig. 2) at a flow rate of 0.02-0.03 ml/min. The radioactivity of fractions collected at 3-h intervals for 15 h, and filters was determined as previously described
187
(Kohn et al., 1981). Because of the greater frequency of DNA single-strand breaks produced by topoisomerase II inhibitors, elutions of cells treated with m-AMSA or VP-16 were performed at higher flow rate (0.04-0.12 ml/min) with internal standard cells irradiated with 2000 rad 7irradiation and fractions were collected at 5-min intervals for 30 min (Zwelling et al., 1981).
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Determination of protein-free DNA single-strand breaks DNA single-strand breaks not associated with protein (frank breaks) were detected by alkaline elution carried out under DNA denaturing, nondeproteinizing conditions as described for the DNA-protein crosslink assay, except that treated cells received no irradiation prior to elution and that SDS lysis solution was used instead of LS10 (Kohn et al., 1981). Elution rates were compared with those from irradiated control cells and considered to arise from DNA fragments not bound to protein. Results
Exposure of normal or Bloom-syndrome fibroblasts to BrdU for 5 days produced DNA single-strand breaks (Figs. 1-3). One micromolar BrdU produced more than 300 rad-equivalents DNA single-strand breaks upon exposure to alkali with both cell types (Fig. 1). In contrast to the case of y-irradiation, the elution curves of cells treated with BrdU displayed a non-linear shape with an increase in slope with elution time. This indicates that DNA cleavage was produced by exposure of the cell lysates to the alkaline elution solution and suggested that BrdU produced alkaline labile sites in DNA. The effect of the elution solution pH upon the elution rate of DNA from cells treated with BrdU was determined. Fig. 2 shows such an experiment performed with normal fibroblasts. In contrast to the breaks produced by ),-irradiation, more DNA breaks were detected at pH 12.6 than 12.1 in BrdU-treated cells. Similar results were obtained with Bloomsyndrome fibroblasts (data not shown). This result confirms that BrdU produced alkali-labile lesions in the DNA. Similar DNA elutions were observed under de-
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FRACTION OF 3H-DNA ON FILTER Fig. 1. DNA single-strand breaks produced by BrdU in normal (closed symbols) and Bloom-syndrome fibroblasts (open symbols). Cells were labeled with [14C]thymidine (0.02 /~Ci/ml) and BrdU for 5 days in the dark. Typical alkaline elution curves are shown. • and O: control cells; • and 13: calibration cells irradiated with 300 rad; • and zx: BrdU 1/tM; • and v: BrdU 10 ttM; 41, and ~: BrdU 10 ttM followed by elution under non-deproteinizing conditions. The DNA breaks were not protein-linked. No difference was observed between Bloom syndrome and normal fibroblasts.
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Fig. 2. Effect of elution pH upon the production of DNA single-strand breaks by BrdU. Normal fibroblasts were labeled and treated with ]/~M BrdU as described in the legend of Fig. 1. Alkaline clution was either performed at pH 12.1 (closed symbols) or 12.6 (open symbols). Circles: control cells; triangles: BrdU-treated cells; and squares: cells irradiated with 300 tad. In contrast to the breaks made by X-rays, the production of DNA single-strand breaks by BrdU was increased upon the addition of alkali.
-
188 proteinizing and non-deproteinizing conditions (Fig. 1). Therefore the D N A breaks produced by BrdU were not protein-linked. This finding eliminates the possibility that the production of D N A breaks by BrdU was mediated by topoisomerases, because with topoisomerases the breaks are protein-associated (Pommier and Kohn, 1988). Since treatment with 1 /*M BrdU results in elution curves which are too rapid to permit detection of a difference between normal and Bloomsyndrome cells (Fig. 1), a comparison of the D N A single-strand break frequencies in the two cell lines was performed over a lower range of BrdU concentrations (Fig. 3). The D N A strand breaking effect of BrdU was concentration-dependent and also detectable at concentrations as low as 0.1/,M. Under these conditions more D N A single-strand breaks were produced in normal than in Bloomsyndrome fibroblasts (Fig. 3). This greater frequency is probably related to a greater D N A incorporation of BrdU into normal cells than into Bloom-syndrome cells since the growth rate of normal fibroblasts was approximately 25% faster than that of Bloom-syndrome fibroblasts. The absence of protein-linkage of the D N A
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Mutation Research, DNA Repair, 254 (1991) 185-190 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100060Y MUTDNA 06423
Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom-syndrome fibroblasts Yves Pommier, Thomas M. Ranger, Donna Kerrigan and Kenneth H. Kraemer Laboratory of Molecular Pharmacology (YP, DK) and Laboratory of Molecular Carcinogenesis (TMR, KK), National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (U.S.A.) (Received 1 March 1990) (Revision received 3 August 1990) (Accepted 10 August 1990)
Keywords: Bloom-syndrome fibroblasts; DNA strand breakage; Bromodeoxyuridine; Topoisomerase II
Smmnary Cells from patients with Bloom syndrome, a cancer-prone disorder with cutaneous photosensitivity and spontaneous chromosome breakage, exhibit an abnormally increased number of sister-chromatid exchanges following treatment with 5-bromodeoxyuridine (BrdU). This effect has been postulated to be mediated by abnormal topoisomerase II activity. We used alkaline elution to measure DNA single-strand breakage following prolonged exposure to BrdU. Five-day exposure to BrdU produced equal numbers of alkali-labile sites in normal and Bloom-syndrome fibroblasts. These breaks were not protein-associated but were produced by alkali. Treatment with topoisomerase II inhibitors induced similar frequencies of DNA single-strand breaks in normal and Bloom-syndrome fibroblasts. These findings imply that BrdU incorporation into cellular DNA induces alkali-labile DNA lesions that are independent of topoisomerase II activity in Bloom and normal cells.
Bloom syndrome is a rare autosomal recessive disease with a high cancer risk, sun sensitivity, immunodeficiency and growth retardation (German, 1969). Cells from patients with Bloom-syndrome are characterized by an elevated level of spontaneous chromosome breakage and sisterchromatid exchanges (SCE). The chromosome breakage and SCE increase upon exposure to 5bromodeoxyuridine (BrdU) (Chaganti et al., 1974; Heartlein et al., 1987; Tsuji et al., 1988). BrdU has
Correspondence: Dr. Yves Pommier, Bldg. 37, Rm. 5C27, National Cancer Institute, Bethesda, MD 20892 (U.S.A.), Telephone: (301)496-5944.
also been reported to produce alkaline-labile DNA strand breaks (Dillehay et al., 1984; Fornace et al., 1986). Because it has been reported that the induction of SCE by antitumor topoisomerase II inhibitors may be related to the trapping of topoisomerase II-mediated DNA breaks (Pommier et al., 1985), it has been suggested that the induction of SCE by BrdU may result from abnormal topoisomerase II activity in Bloom-syndrome cells (Heartlein et al., 1987). Other reports suggested a DNA ligase I deficiency in Bloom-syndrome cells (Willis et al., 1987; Chan et al., 1987; Ranger and Kraemer, 1989) and abnormal antigenicity of uracil DNA glycosylase (Volberg et al., 1987). We examined the role of DNA topoisomerase
186
II in BrdU-induced DNA breakage in Bloom-syndrome and normal fibroblasts. DNA topoisomerase II binds to DNA, produces a double-strand break while covalently binding to DNA termini and then rejoins the ends of the DNA following a topological unwinding (Wang, 1987; Pommier and Kohn, 1988; Liu, 1989). Topoisomerase II-induced DNA breaks are characteristically present under deproteinizing conditions and are not detectable under non-deproteinizing conditions (Pommier and Kohn, 1988; Covey et al., 1989). Antitumor topoisomerase II inhibitors, such as amsacrine (m-AMSA) and teniposide (VP-16) function by trapping the topoisomerase II-DNA complex after the DNA has been cut by the topoisomerase II but prior to the rejoining of the cut ends (Wang, 1987; Pommier and Kohn, 1988; Liu, 1989). The frequency of protein-linked DNA breaks following treatment with topoisomerase II inhibitors thus is a measure of the extent of topoisomerase II activity within the cells. In the present study we used alkaline elution to measure the induction of protein-linked and nonprotein-linked DNA breaks by BrdU in Bloomsyndrome and normal cells. We also assessed the topoisomerase II activity in these cells following treatment with topoisomerase II inhibitors.
Mutant Cell Repository, Coriel Institute for Medical Research, Camden, NJ. Cells were grown in monolayer cultures in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum and 20 mM glutamine in a humidified atmosphere with 8% CO 2. Fibroblast doubling times were approx. 30 h (normal) and 40 h (Bloom syndrome). Suspension cultures of L1210 leukemia cells were maintained as previously described (Kohn et al., 1981). For alkaline elution experiments, normal and Bloom-syndrome fibroblast cell lines were plated at 1 × 105 cells/25 cm2 in medium containing 0.02 /~Ci/ml [14C]thymidine and grown for 2-5 days. L1210 cells used for internal standards were labeled for 16-24 h with 0.2 /~Ci/ml [3H]thymidine and chased in unlabeled medium for at least 2 h prior to use. In the BrdU experiments cells were grown in the presence of both BrdU and 10/~M FdU for 5 days (Heartlein et al., 1987) and maintained in the dark throughout the experiments. Topoisomerase inhibitors (m-AMSA, VP-16) were added to cell cultures such that the concentration of DMSO never exceeded 0.25%. Drug exposures were for 30 rain at 37°C, and drug treatments were terminated by rinsing cells twice with ice-cold Hanks' Balanced Salt Solution (HBSS) and then scraping them into the same buffer.
Materials and methods
Chemicals 5-Bromodeoxyuridine (BrdU) and fluorodeoxyuridine (FdU) were obtained from Sigma Chemical Co., St. Louis, MO. [Me-3H]Thymidine and [2-14C]thymidine (spec. act. 20 and 0.05 /~Ci/mmole, respectively), were purchased from NEN Research Products (Boston, MA). 4'-(9Acridinylamino)methanesulfon-m-anisidide (mAMSA) (NSC 249992) was obtained from the Pharmaceutical Resources Branch, Division of Cancer Treatment, NCI, Bethesda, MD. Etoposide (VP-16) was a gift from the Bristol Myers Co., Wallingford, MA. Stock solutions were prepared in dimethyl sulfoxide (DMSO) at 10 mM. Cell cultures, and BrdU treatment Simian virus 40 (SV40) transformed fibroblasts (normal: GM 00637; and Bloom syndrome: GM 08505A) were obtained from the Human Genetic
Determination of DNA single-strand break frequency DNA single-strand breaks were analyzed using alkaline elution carried out under deproteinizing conditions as described previously (Kohn et al., 1981). Briefly, aliquots of [14C]-labeled cells were mixed in ice-cold phosphate-buffered saline (PBS) with [3H]-labeled internal standard cells that had been irradiated with 300 rad ~,-radiation immediately before. Cells were then layered onto polycarbonate filters (2.0 /~m pore diameter, Nucleopore Corp., Pleasanton, CA) and were lysed with a solution containing 2% SDS, 0.1 M glycine, 0.025 M Na2EDTA, and 0.5 mg/ml proteinase K, pH 10 (SDS-Lysis). The lysis solution was washed with 5 ml of 0.02 M EDTA, pH 10, and the DNA was eluted with tetrapropylammonium hydroxide-EDTA, 0.1% SDS, pH 12.1 (or 12.6; Fig. 2) at a flow rate of 0.02-0.03 ml/min. The radioactivity of fractions collected at 3-h intervals for 15 h, and filters was determined as previously described
187
(Kohn et al., 1981). Because of the greater frequency of DNA single-strand breaks produced by topoisomerase II inhibitors, elutions of cells treated with m-AMSA or VP-16 were performed at higher flow rate (0.04-0.12 ml/min) with internal standard cells irradiated with 2000 rad 7irradiation and fractions were collected at 5-min intervals for 30 min (Zwelling et al., 1981).
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Determination of protein-free DNA single-strand breaks DNA single-strand breaks not associated with protein (frank breaks) were detected by alkaline elution carried out under DNA denaturing, nondeproteinizing conditions as described for the DNA-protein crosslink assay, except that treated cells received no irradiation prior to elution and that SDS lysis solution was used instead of LS10 (Kohn et al., 1981). Elution rates were compared with those from irradiated control cells and considered to arise from DNA fragments not bound to protein. Results
Exposure of normal or Bloom-syndrome fibroblasts to BrdU for 5 days produced DNA single-strand breaks (Figs. 1-3). One micromolar BrdU produced more than 300 rad-equivalents DNA single-strand breaks upon exposure to alkali with both cell types (Fig. 1). In contrast to the case of y-irradiation, the elution curves of cells treated with BrdU displayed a non-linear shape with an increase in slope with elution time. This indicates that DNA cleavage was produced by exposure of the cell lysates to the alkaline elution solution and suggested that BrdU produced alkaline labile sites in DNA. The effect of the elution solution pH upon the elution rate of DNA from cells treated with BrdU was determined. Fig. 2 shows such an experiment performed with normal fibroblasts. In contrast to the breaks produced by ),-irradiation, more DNA breaks were detected at pH 12.6 than 12.1 in BrdU-treated cells. Similar results were obtained with Bloomsyndrome fibroblasts (data not shown). This result confirms that BrdU produced alkali-labile lesions in the DNA. Similar DNA elutions were observed under de-
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FRACTION OF 3H-DNA ON FILTER Fig. 1. DNA single-strand breaks produced by BrdU in normal (closed symbols) and Bloom-syndrome fibroblasts (open symbols). Cells were labeled with [14C]thymidine (0.02 /~Ci/ml) and BrdU for 5 days in the dark. Typical alkaline elution curves are shown. • and O: control cells; • and 13: calibration cells irradiated with 300 rad; • and zx: BrdU 1/tM; • and v: BrdU 10 ttM; 41, and ~: BrdU 10 ttM followed by elution under non-deproteinizing conditions. The DNA breaks were not protein-linked. No difference was observed between Bloom syndrome and normal fibroblasts.
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Fig. 2. Effect of elution pH upon the production of DNA single-strand breaks by BrdU. Normal fibroblasts were labeled and treated with ]/~M BrdU as described in the legend of Fig. 1. Alkaline clution was either performed at pH 12.1 (closed symbols) or 12.6 (open symbols). Circles: control cells; triangles: BrdU-treated cells; and squares: cells irradiated with 300 tad. In contrast to the breaks made by X-rays, the production of DNA single-strand breaks by BrdU was increased upon the addition of alkali.
-
188 proteinizing and non-deproteinizing conditions (Fig. 1). Therefore the D N A breaks produced by BrdU were not protein-linked. This finding eliminates the possibility that the production of D N A breaks by BrdU was mediated by topoisomerases, because with topoisomerases the breaks are protein-associated (Pommier and Kohn, 1988). Since treatment with 1 /*M BrdU results in elution curves which are too rapid to permit detection of a difference between normal and Bloomsyndrome cells (Fig. 1), a comparison of the D N A single-strand break frequencies in the two cell lines was performed over a lower range of BrdU concentrations (Fig. 3). The D N A strand breaking effect of BrdU was concentration-dependent and also detectable at concentrations as low as 0.1/,M. Under these conditions more D N A single-strand breaks were produced in normal than in Bloomsyndrome fibroblasts (Fig. 3). This greater frequency is probably related to a greater D N A incorporation of BrdU into normal cells than into Bloom-syndrome cells since the growth rate of normal fibroblasts was approximately 25% faster than that of Bloom-syndrome fibroblasts. The absence of protein-linkage of the D N A
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