Cell Cycle-dependent Cytotoxicity of Alkylating Agents: Determination of Nitrogen. Mustard-induced DNA Cross-Links and Their Repair in Chinese Hamster.
[CANCER RESEARCH 46, 2324-2329, May 1986]
Cell Cycle-dependent Cytotoxicity of Alkylating Agents: Determination of Nitrogen Mustard-induced DNA Cross-Links and Their Repair in Chinese Hamster Ovary Cells Synchronized by Centrifugal Elutriation1 David Murray2 and Raymond E. Meyn Department of Experimental Radiotherapy, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77030
ABSTRACT Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal vlut ria t¡onand treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antituntor agent. In agreement with previous studies, cell populations enriched in 2-95%air atmosphere in McCoy's 5A medium (Hsu's modification, Formula No. 78-5192; GIBCO, Grand Island, NY) supplemented with 15% FBS (Irvine Sci entific, Santa Ana, CA). Under these conditions, the cells had a 12- to 14 h doubling time and an 11.5-h generation time; the approximate durations of the individual phases were 2.5 (d), 6.75 (S), 1.75 (G2), and 0.5 h (M). Prior to elutriation, cells were harvested at 37°Cusing
0.025% trypsin containing DNase, 20 Mfi/nil. Trypsinization was quenched after 7 min by adding an equal volume of complete medium, and the cells were vigorously pipetted and syringed through a 22-gauge needle to minimize clumping. Light microscopy was used to inspect the quality of the single-cell suspensions. Centrifugal Elutriation. Cells were synchronized by the centrifugal Received 4/15/85; revised 1/15/86; accepted 2/3/86. elutriation method (9, 10), which exploits the fact that cells continu 1This investigation was supported by USPHS grant CA 23270 awarded by the ously increase in size as they progress through the cell cycle. This National Cancer Institute. 2 To whom requests for reprints should be addressed, at Department of method has several advantages (9) including the fact that it yields Experimental Radiotherapy, Box 66, M. D. Anderson Hospital and Tumor adequate synchrony for this application when compared with other Institute, 6723 Bertner Avenue, Houston TX 77030. techniques (10, 11). One disadvantage is that it does not allow charac 3The abbreviations used are: CHO, Chinese hamster ovary; HN2, nitrogen terization of M cells independently of 62 cells. The centrifuge (Model mustard; PSA, Puck's solution A: CLF, cross-link factor, GSH, reduced glutaJ-21C fitted with a Model .IF 6 elutriator rotor, Beckman Instruments, thione; FBS, fetal bovine serum; MEM, minimum essential medium; SDS, Palo Alto, CA) was operated at a constant rotor speed of 1700 rpm. sodium dodecyl sulfate. 2324
CELL CYCLE DEPENDENCE
Between 1 x 10* and 2.5 x 10" cells suspended in 20 ml of medium were introduced into the elutnation chamber at a flow rate of 12 ml/ min and elutriated at room temperature by increasing the flow rate of medium (a-MEM containing 5% FBS and DNase, 20 Mg/ml) through the chamber in a stepwise manner from about 12 to about 30 ml/min. In all, twelve SO-mlfractions were collected and placed on ice. Aliquots were withdrawn for cell count and Coulter volume determinations, and another aliquot was fixed (in 1 ml saline:3 ml 70% ethanol) for analysis by flow cytometry to determine the purity of the individual fractions using an ICP-21 flow cytome ter (Ortho Instruments, Westwood, MA). The proportion of cells of each phase in a particular fraction was determined as described by Johnston et al. (12). Drug Treatments. Either asynchronous cells or cell fractions syn chronized by centrifugal elutriation were sedimented by centrifugation, resuspended in medium (containing 20% FBS) at a density of about 1.3 x 11)"/ml.and plated onto 60-mm tissue culture dishes to give about 2 x 10*cells/dish. After incubation for 30 min at 37*C almost complete attachment of the cells was observed. The cells were then washed twice with PSA and exposed to various concentrations of HN2 in serum-free a-MEM for 0.5 or l h at 37'C. Stock solutions of HN2 (mechlorethamine hydrochloride; Merck, Sharp, and Dohme, West Point, PA) were stored frozen in physiological saline at a concentration of 100 pg/ml. Aliquots were thawed for 0.5 h at room temperature just before use and then diluted to the required concentration with serum-free a-MEM. After treatment, the cells were washed twice with PSA and either trypsinized immediately (0.025% trypsin) for 7 min at 37*C (cells for cytotoxicity or initial DNA damage determination) or were incubated at 37*C in complete McCoy's medium for periods up to 6 h before trypsinization to allow for repair of the H N2-induced DNA cross-links. Cytotoxicity was assessed by colony-forming ability, surviving cells being identified by their ability to form colonies of 50 cells or more. Alkaline Elution. DNA cross-linking was estimated using the alkaline elution methodology originally developed by Kohn (8), with slight modification (6). Briefly, cells were labeled overnight with [MC]thymi-
OF DNA CROSS-LINKING
linear regression analysis. The error limits associated with the slope values represent the 95% confidence interval. Any DNA strand breaks induced by the drug treatment would be detected as an increased rate of elution in the alkaline elution profiles obtained from 11N2 treated unirradiated control samples that were run simultaneously with the 4 Gy-irradiated samples. Glutathione. GSH was determined as follows. Various numbers (be tween 0 and 10 x IO6) of cells were pelleted by centrifugation (2000 rpm for 10 min at 25*Q, washed by resedimenting from PSA, and then resuspended in 4 ml of distilled water. After vortexing the suspension for 5 min, metaphosphoric acid (1 ml, 25%) was added, and after a further 5 min of vortexing, cell debris was removed by centrifugation (3000 rpm for 20 min) followed by passage of the supernatant through a 0.45-/»mMillex filter (Millipore Corp., Bedford, MA). GSH in the supernatant was estimated using the fluorescent dye 0-phthalicdicarboxaldehyde (15). The relative fluorescence emission was a linear function of the number of cells pelleted within this range but flattened out somewhat at higher cell numbers (data not shown). The fluorescence of the GSH standard solutions was also linear over the range 0-0.05 HIM.GSH concentrations were determined from the relationship
GSH = -- x 10-" mol/cell ¿nr where n was the number (in millions) of cells assayed, /was the sample fluorescence, and F was the fluorescence from a standard 0.01 HIM GSH solution.
RESULTS Cytotoxicity The cell cycle phase dependence of HN2 cytotoxicity deter mined after exposure of cells from each elutriator fraction to a single dose (3 ng/m\ for 1 h) of HN2 is shown in Fig. 1. Cells in G] were the most sensitive, followed by cells at the GrSphase transition; cells in mid to late S phase tended to be the most resistant. Cells varied in sensitivity by a factor of about
dine (0.01 /iCi/ml; 50 mCi/mmol; Schwarz/Mann, Orangeburg, NY) and then chased for 8 h with label-free medium. After drug treatment and trypsinization, an appropriate volume to give 8 x 10s cells was impinged onto a 25-mm polycarbonate membrane (2-pm pore; Nudepore Corp., Pleasanton, CA). The cells were then rinsed twice with icecold PSA containing 5 HIMEDTA, lysed with 5 ml of SDS lysis solution (0.025 M EDTA-2% SDS, final pH 9.7) and then rinsed with 0.1 5 ml of 0.02 M EDTA (pH 10.3). The DNA was eluted overnight in the dark with 0.1 M tetrapropyl ammonium hydroxide-)1.02 M EDTA (free acid)-0.1% SDS (final pH 12.15) at a constant flow rate of 0.04 ml/min to give 10 equal fractions of about 3.5 ml. The DNA in the resulting fractions and that remaining both on the membrane and on the interior of the membrane holder at the completion of the elution experiment was measured by liquid scintillation counting (6). The rate of elution of DNA from HN2-treated cells from the membrane may be influenced by both DNA-interstrand and DNA-protein cross-links (13). Although the use of SDS lysis and elution solutions combined with polycarbonate membranes served to minimize the effect of DNAprotein cross-links on the elution kinetics (14), additional experiments 0.001were performed in which proteinase K (0.5 mg/ml) was added to the lysis solution and retained in contact with the membrane for 0.5 h prior to the elution of the DNA. This treatment further reduces the influence of DNA-linked protein molecules on the elution rate. Those lesions resistant to digestion are presumed to be predominantly DNA-inter strand cross-links, whereas those removed by the proteinase K are 0.0001 presumed to represent DNA-protein cross-links (13, 14). 14 12 10 8 Determination of DNA Cross-Linking. To determine DNA crossFraction Number linking, the cell suspensions were irradiated on ice with 4 Gy of X-rays Fig. 1. Age-response pattern for Chinese hamster ovary cells exposed to a immediately prior to the alkaline elution to introduce a constant num ber of DNA strand breaks (6). CLFs were calculated from the ratio of single concentration (3.0 Mg/ml) of HN2. Cells were separated into fractions enriched in different phases of the cell cycle by centrifugal elutriation and then the log of the fraction of uneluted DNA for the 4-Gy-irradiated control treated with HN2 (l h at 37*C) in medium supplemented with 15% fetal bovine to that for the 4-Gy-irradiated UN2-treated sample, both values being serum. Following treatment, the cells were plated out for colony formation. The determined at the same eluted volume (21 ml in this case, although the early fractions (4-5) represent cells enriched in d (e.g., fraction 4 was 96% d, 3% S phase, 1%G2 + M); middle fractions (6-10) were enriched in S phase (e.g., relative CLFs did not depend greatly on the actual volume selected). fraction 8 was 30% G,, 68% S phase, 2%
