Induces Thymineless Death with Topoisomerase I-DNA Complexes
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Research Article A Novel Polypyrimidine Antitumor Agent FdUMP[10] Induces Thymineless Death with Topoisomerase I-DNA Complexes Zhi-Yong Liao,1 Olivier Sordet,1 Hong-Liang Zhang,1 Glenda Kohlhagen,1 Smitha Antony,1 William H. Gmeiner,2 and Yves Pommier1 1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, NIH, Department of Health and Human Services, Bethesda, Maryland and 2Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, North Carolina Abstract with camptothecins. Because camptothecin and its derivatives FdUMP[10], a 10mer of 5-fluoro-2V-deoxyuridine 5V-monophos- selectively trap topoisomerase I (Top1; refs. 12, 13), in this report, phate (FdUMP), the thymidylate synthase inhibitory metabo- we studied the possible relationships between Top1 inhibition and lite of 5-fluorouracil (FU), is most closely correlated with the the cellular mechanisms of action of FdUMP[10]. DNA topoisomerase I (Top1) inhibitor camptothecin in the Mammalian Top1 is an essential enzyme, relaxing DNA super- National Cancer Institute COMPARE analysis, but not with FU. coiling ahead of replication and transcription (14, 15). To relax FdUMP[10] exhibits more potent antiproliferative activity DNA, Top1 reversibly creates transient single-strand breaks by V than FdUMP or 5-fluoro-2V-deoxyuridine (FdU) and is mark- forming covalent tyrosyl-phosphodiester bonds with the 3 ends of edly more active than FU. Camptothecin-resistant P388/CPT45 the broken DNA. Once the DNA is relaxed, Top1 readily relegates the breaks and regenerates intact duplex DNA. Under normal cells lacking Top1 are cross-resistant to FdUMP[10] as well as to FdUMP, FdU, and the thymidylate synthase inhibitor physiologic conditions, these intermediates, referred to as the raltitrexed (Tomudex). FdUMP[10] induces DNA single-strand ‘‘Top1 cleavage complexes’’ are very transient: the religation step of breaks and cellular Top1-DNA complexes. Such complexes are the DNA cleavage/religation equilibrium is favored and only a small also observed in response to FdUMP, FdU, raltitrexed, and FU. fraction of the DNA is cleaved at any given time (12, 13, 16). Top1 The FdUMP[10]-induced Top1-DNA complexes are not inhibitors, such as camptothecins, trap the cleavage complexes by inhibited by the caspase inhibitor z-VAD-fmk and form inhibiting the religation step (13, 16, 17). Top1 cleavage complexes independently of apoptotic DNA fragmentation, indicating can also be trapped by a wide range of DNA alterations, including that they do not correspond to apoptotic Top1-DNA com- uracil misincorporations, base mismatches, abasic sites, nicks, plexes. In biochemical assay, Top1 is directly trapped at uracil oxidized bases, UV photo-lesions, and carcinogenic adducts (18, 19). and FdU misincorporation sites. We propose that FdUMP[10] Top1-DNA complexes also occur during apoptosis in response to damages DNA by trapping Top1 at uracil and FdU misincor- oxygen radicals and mitochondrial dysfunction (20–22). poration sites resulting from thymidylate synthase inhibition In this report, we show high COMPARE correlation between FdUMP[10] and several camptothecin derivatives, cross-resistance of and thymine depletion. (Cancer Res 2005; 65(11): 4844-51) Top1-deficient cells to FdUMP[10], FdUMP, 5-fluoro-2V-deoxyuridine (FdU), and raltitrexed, and induction of Top1-DNA cleavage Introduction complexes in mouse and human cancer cells treated with 5-Fluorouracil (FU) was developed in the late 1950s (1) as an FdUMP[10], FdUMP, FdU, and raltitrexed. We also show induction antimetabolite inhibiting RNA and DNA biosynthesis. FU and other of Top1 cleavage complexes by FdU incorporation in DNA fluoropyrimidines have been widely used for the treatment of oligonucleotides. A model is presented to explain Top1 trapping human cancers, including colorectal, breast, and head and neck by FdUMP[10] treatment and thymidine depletion. cancers for more than 40 years (2). The mechanism of action of FU has been ascribed to the inhibition of thymidylate synthase by its Materials and Methods active metabolite 5-fluoro-2V-deoxyuridine 5V-monophosphate (FdUMP; ref. 3), induction of ‘‘thymineless death’’ (4, 5), and RNA Chemicals and enzymes. Camptothecin was provided by M.E. Wall synthesis inhibition (6). (Research Triangle Institute, Research Triangle Park, NC). FU, FdU, and FdUMP were from Sigma (St. Louis, MO) and raltitrexed was a gift from In the past decades, significant research effort has continued to Dr. Godefridus J. Peters (VU University, Amsterdam, the Netherlands). focus on developing strategies to enhance the antineoplastic FdUMP[10] was prepared in Dr. William H. Gmeiner’s laboratory. activity of FU through biochemical modulation and different [3H]Thymidine and [14C]thymidine were purchased from Perkin-Elmer Life methods of administration (2, 7). More recently, FdUMP[10], a Sciences (Boston, MA). [32P]Cordycepin 5V-triphosphate and terminal 10mer of FdUMP, was developed (8–11; Fig. 1). FdUMP[10] is 338- deoxynucleotidyl transferase were purchased from DuPont-New England fold more potent than FU at inhibiting cell proliferation in the Nuclear (NEN, Boston, MA) and Life Technologies (Gaithersburg, MD). National Cancer Institute (NCI) 60 cell line screen (11). Unexpect- Human recombinant Top1 was purified as described (23). edly, COMPARE analysis showed that the FdUMP[10] activity Cell lines and cultures. Mouse leukemia P388 and P388/CPT45 cells profile clearly differed from FU but was most closely correlated were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% heat- inactivated FCS (Invitrogen) and 10 Amol/L h-mercaptoethanol (21, 22, 24, 25). Human leukemia CEM cells and colorectal carcinoma HCT116 cells were cultured in DMEM containing 10% FCS. The human colon cancer Requests for reprints: Yves Pommier, Building 37, Room 5066, Laboratory of HCT-C18 cell line was a kind gift from Dr. Edward Chu at Yale University Molecular Pharmacology, 37 Convent Drive, NIH, Bethesda, MD 20892-4255. Phone: 301-496-5944; Fax: 301-402-0752; E-mail: [email protected]. (New Haven, CT) and were maintained in RPMI 1640 containing 10% FCS I2005 American Association for Cancer Research. supplemented with 10 Amol/L thymidine (26). The HCT-C:His-TS(+) cell Cancer Res 2005; 65: (11). June 1, 2005 4844 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2005 American Association for Cancer Research. FdUMP[10] Thymineless Death and Top1 Figure 1. Chemical structures of FdUMP[10] and other compounds most closely related to FdUMP[10] in COMPARE analysis (see Table 1). Numbers in bold, drug rank number in COMPARE analysis. line was established by stable transfection of HCT-C18 cells with human [3H]Thymidine incorporation (percent control) was calculated as the ratio of His-tagged thymidylate synthase cDNA (27). Murine FM3A/0 and FM3A/ [3H]:[14C] in the treated samples over that in the untreated samples. TKÀ mammary carcinoma cells were a gift from Dr. Godefridus J. Peters Effect of 5-fluoro-2V-deoxyuridine on topoisomerase I cleavage (28). FM3A/0 and FM3A/TKÀ cells were maintained in RPMI 1640 complexes. We designed model oligonucleotides containing one or two containing 10% fetal bovine serum. FdU incorporated immediately 3V from a preexisting Top1 cleavage site 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (33, 34) to study the effect of FdU incorporation on the Top1-DNA assays for drug cytotoxicity. Drug cytotoxicity was measured using cleavage complexes (see Fig. 7A). Oligonucleotides were purchased from standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MWG Biotech Co. (High Point, NC). 3V Labeling of single-stranded (MTT) assay after 72 hours drug exposure. The absorbance was measured oligonucleotides and annealing were done as described (33, 34). Duplex at 560 nm using a Bio-Tek Microplate Reader (Molecular Devices, Palo DNA substrates were generated by annealing a 3V-labeled upper strand 32 Alto, CA). [5V-AAAAAGACTTGGAAAAATTTT*-3V, where * corresponds to the a- P- DNA single-strand break induction by FdUMP[10]. DNA single-strand labeled cordycepin] with three types of FdU (F) modified 22mer lower breaks were assayed by alkaline elution according to published protocols strands (FdU + 1, FdU + 2, and FdU + 1 + 2; see Fig. 7A). Top1 reactions (29, 30). were done as described (33, 34). Detection of covalent topoisomerase I-DNA complexes in cells. Top1-DNA complexes were isolated using the immunocomplex of enzyme Results (ICE) bioassay (25, 31, 32). Briefly, 106 cells were lysed with 1 mL of 1% sarkosyl. After Dounce homogenization, cell lysate was layered on cesium Relationship between topoisomerase I and antiproliferative chloride step gradients and centrifuged at 165,000 Â g for 20 hours at 20jC. activity of FdUMP[10]. In the 60 cell lines from the NCI Fractions (500 AL) were collected from the bottom, diluted with an equal Developmental Therapeutics Program (DTP), FdUMP[10] [National volume of 25 mmol/L sodium phosphate buffer (pH 6.6), and applied to Service Center (NSC) 697912] is 338-fold more effective than FU Immobilon-P membranes (Millipore, Bedford, MA) by using a slot-blot (NSC 19893; based on mean GI50 values; ref. 11). COMPARE analysis vacuum manifold. Top1-DNA adducts were detected by immunoblotting (http://dtp.nci.nih.gov; ref. 35) was used to reveal the potential using the C21 mouse monoclonal Top1 antibody obtained from Dr. Yung-Chi mechanism of action of FdUMP[10]. Table 1 shows the Pearson Cheng (Yale University). correlation coefficients for the compounds with the highest Incorporation of thymidine into DNA. HCT116 cells in exponential correlation, as well as FU, which ranked 237th among all the 14 growth phase were first labeled with 0.025 ACi/mL of [ C]thymidine for compounds/extracts deposited in the DTP database. The best j 48 hours at 37 C. After FdUMP[10] treatment for 48 hours, cells were pulse- correlated compounds, besides FdUMP[10], chosen as the seed, labeled with 1 ACi/mL of [3H]thymidine for 10 minutes at 37jC. After washing and therefore has a Pearson correlation coefficient of 1, include the cells twice with cold HBSS, cells were scraped in 4 mL of cold HBSS.