The DNA Minor Groove-alkylating Cyclopropylpyrroloindole Drugs Adozelesin and Bizelesin Induce Different DNA Damage Response Pathways in Human Colon Carcinoma HCT116 Cells 1 Pei-rang Cao, 2 Mary M. McHugh, 2 Thomas Melendy, and Terry Beerman 3 Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263 [P-r. C., M. M. M., T. B.], and Department of Microbiology and the Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, School of Medicine and Biomedical Sciences, Buffalo, New York 14214 [T. M.] Abstract As members of the cyclopropylpyrroloindole family, adozelesin and bizelesin cause genomic DNA lesions by alkylating DNA. Adozelesin induces single-strand DNA lesions, whereas bizelesin induces both single-strand lesions and double-strand DNA cross-links. At equivalent cytotoxic concentrations, these agents caused different biological responses. Low adozelesin concentrations (e.g., 0.5 nM) induced a transient S-phase block and cell cycle arrest in G 2 -M, as well as increased induction of p53 and p21, whereas a high drug concentration (e.g., 2.5 nM) caused apoptosis but no p21 induction. In contrast, both low and high bizelesin concentrations enhanced p53 and p21 induction and triggered G 2 -M cell cycle arrest and eventual senescence without significant apoptotic cell death. However, in cells lacking p21, bizelesin, as well as adozelesin, triggered apoptosis, indicating that p21 was crucial to sustained bizelesin- induced G 2 -M arrest. Thus, despite similar abilities to alkylate DNA, the chemotherapeutic agents adozelesin and bizelesin caused a decrease in HCT116 tumor cell proliferation by different pathways (i.e., adozelesin induced apoptosis, and bizelesin induced senescence). Introduction Adozelesin and bizelesin (see Fig. 1), members of the CPI 4 family, alkylate in the minor groove of DNA at A-T rich regions (1). Adozelesin is a monofunctional CPI that alkylates at a single adenine, causing a single-strand DNA lesion (1). In contrast, bizelesin is a CPI dimer with two reactive chloro- methyl moieties that can form DNA adducts with adenines in either one or both DNA strands, leading to single-strand DNA lesions or double-strand DNA cross-links, respectively (2). When compared with other therapeutic agents that inhibit DNA synthesis by alkylating genomic DNA [e.g., methylmeth- ane sulfonate and cisplatin (3)], adozelesin and bizelesin are extraordinarily cytotoxic. They inhibit growth of a variety of murine and human tumor xenografts without the lethal hep- atotoxicity caused by the parent compound CC-1065 (4, 5). Both adozelesin and bizelesin have undergone clinical trials (6). Although they alkylate DNA with similar sequence prefer- ences, the damage induced by adozelesin and bizelesin affects different biological responses. Bizelesin is more cy- totoxic than adozelesin to both normal and tumor cells (7). Also, although both adozelesin and bizelesin block initiation of DNA synthesis in intact cells (8), adozelesin is severalfold more potent than bizelesin when DNA synthesis inhibition is measured either in intact cells or in a cell-free replication assay (8). Additionally, whereas adozelesin inhibits DNA rep- lication by causing a decrease in functional RPA (9), bizelesin inhibits replication through induction of a replication inhibitor (8). The mechanisms involved in these differences are largely unknown. Although the effect of bizelesin on the cell cycle has not been reported, adozelesin is known to trigger a transient slowing in cell cycle progression through S phase and arrest in G 2 -M (4). Such a cell cycle response is observed with various types of DNA damage (10). Cells are transiently ar- rested in G 1 , S, or G 2 -M to allow time for DNA repair and to minimize the replication and segregation of damaged DNA (11). p53 plays an important role in the complex network of signals leading to arrest in different parts of the cell cycle (11). The G 1 -S checkpoint is in part dependent on the p53- regulated transcription of p21, a potent inhibitor of the cyclin- CDK complex required for G 1 -S transition (11). Likewise, p53 is involved in G 2 -M arrest by its regulation of genes such as p21 and 14-3-3- (12), which allows activation of the Cdc2- cyclin B1 complex and reduced entry into mitosis (13). In contrast to the G 1 -S and G 2 -M checkpoints, the S- phase checkpoint, which serves to prevent replication from occurring before repair is completed, is generally believed to be p53 independent (14). Whereas treatment during S-phase with DNA-damaging agents that specifically block DNA rep- lication [e.g., UV radiation (15) and hydroxyurea and aphidi- colin (16)] leads to p53 accumulation and phosphorylation, Received 1/21/03; revised 5/16/03; accepted 5/20/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cate this fact. 1 Supported by NIH Grants CA 77491 and CA 16056 (to T. A. B.) and NIH Grants CA89259 and AI01686 (to T. M.). 2 Both authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-3443; Fax: (716) 845-1575; E-mail: terry.beerman@roswellpark.org. 4 The abbreviations used are: CPI, cyclopropylpyrroloindole; RPA, repli- cation protein A; CDK, cyclin-dependent kinase; PARP, poly(ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. 651 Vol. 2, 651– 659, July 2003 Molecular Cancer Therapeutics on October 3, 2021. © 2003 American Association for Cancer Research. mct.aacrjournals.org Downloaded from