Enhanced 7-Ethyl-10-hydroxycamptothecin (SN-38) Lethality by Methylselenocysteine Is Associated with Chk2 Phosphorylation at Threonine-68 and Down-Regulation of Cdc6 Expression

Methylselenocysteine (MSC) is an organic selenium compound in preventative clinical trials involving prostate, lung, and colon carcinoma. We found that methioninase-activated MSC potentiates 7-ethyl-10-hydroxycamptothecin (SN-38)-induced cell lethality in vitro in the p53-defective human head and neck carcinoma A253 cells. Activated MSC increases chk2 phosphorylation at threonine-68 induced by SN-38, with no significant effect on chk1 phosphorylation. Cell cycle arrest induced by SN-38, however, was not abrogated or potentiated by MSC. These results suggest that the enhanced cellular lethality of SN-38 by MSC was not associated with cell cycle regulation pathways. Because chk2, in addition to its role in cell cycle arrest, can induce apoptosis by phosphorylation/activation, we examined whether increased chk2 phosphorylation could induce preapoptotic DNA fragmentation. DNA damage analysis showed that megabase DNA fragmentation is decreased, accompanied by the increased 30 to 300 kilobase pairs of DNA fragmentation after exposure to SN-38 with MSC, compared with SN-38 alone. No significant changes in the amount of DNA fragments were observed in cells treated with SN-38 or MSC alone. Moreover, proteolytic destruction of DNA replication-associated proteins cdc6, MCM2, and cdc25A may induce a DNA damage checkpoint response. The observed downregulation of DNA replication proteins cdc6, MCM2, and cdc25A after exposure to SN-38 with MSC further indicates a relationship between drug response and DNA damage. Exposure to SN-38 with MSC resulted in a significant increase of poly(ADP-ribose) polymerasecleavage and caspase 3 activation. All together, the data support the hypothesis that enhanced lethality of this combination is associated with increased chk2 phosphorylation at Thr68 and down-regulation of specific DNA replication-associated proteins, which result in poly(ADP-ribose) polymerase cleavage, caspase 3 activation, and the induction of 30 to 300 kilobase pairs of DNA fragmentation. Selenium is an essential dietary trace element that plays an important role in a number of biological functions. Recent studies on selenium have focused on its chemopreventive activity (Ganther, 1999). Our experimental studies in cell cultures and animal models demonstrated that MSC can potentiate cell growth inhibition in vitro and antitumor activity in vivo, induced by topoisomerase I (topo I) poisons in the p53-defective human head and neck carcinoma A253 cells. Although several mechanisms have been proposed for enhancing SN-38 cytotoxicity by MSC, the effects on abrogation of cell cycle arrest and apoptosis induction are considered the most feasible mechanism (Yu et al., 2002). The data from our lab first demonstrated that a better prediction of sensitivity to topo I poisons may be obtained by the characterization of chk1 or chk2 cell cycle checkpoint pathways (Yin et al., 2000). A number of references directly linking chk1 inhibition to checkpoint abrogation would be expected (Hirose et al., 2001, Yu et al., 2002; Kohn et al., 2003). Our data also indicate that the chk1 signaling pathways that mediate cell cycle checkpoints are associated with cellular resistance to topo I poisons in the drug-resistant cell line (Yin et al., 2002). All the data have shown that cell cycle checkpoint regulation pathways are associated with drug sensitivity and resistance to topo I poisons. Thus, we propose This work was supported in part by project grant CA65761 and a Comprehensive Cancer Center Support grant CA16056 from the National Cancer Institute. ABBREVIATIONS: topo, topoisomerase; SN-38, 7-ethyl-10-hydroxycamptothecin; SRB, sulforhodamine B; MSC, Se methylselenocysteine; METase, L-methionine-deamino-mercaptomethane lyase (also known as methioninase); PI, propidium iodide; HBSS, Hanks’ balanced salt solution; PFGE, pulsed-field gel electrophoresis; TBE, Tris-borate/EDTA; SeMET, seleno-L-methionine; kb, kilobase(s); PARP, poly(ADP-ribose) polymerase; PML, promyelocytic leukemia gene; chk, checkpoint kinase; MCM, minichromosome maintenance. 0026-895X/04/6601-153–160$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 1 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3105/11260474 Mol Pharmacol 66:153–160, 2004 Printed in U.S.A. 153 at A PE T Jornals on Sptem er 0, 2017 m oharm . aspeurnals.org D ow nladed from that the potentiated drug sensitivity observed with MSC administered before SN-38 is associated with alteration of specific biochemical and molecular markers associated with chk1 and chk2 cell cycle regulation pathways. Recent experiments (Yu et al., 2001; Sordet et al., 2003) indicated that reduced expression of chk2/hcds1 might promote a p53-independent apoptotic response. In addition, antisense inhibition of chk2/hcds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in human embryonic kidney 293 cells. These recent data provide the evidence that deregulation of chk2/ hcds1 in p53-null cells contributes to enhanced apoptosis. These authors also suggest that the DNA-damage regulating kinase chk2, in addition to its role in cell cycle arrest and/or DNA repair, can induce apoptosis by phosphorylation/activation. Thus, chk2 expression and phosphorylation may play an important role in regulating cell death. Origin licensing proteins cdc6, preRC, and MCM2, play important roles in regulating cell death, in addition to regulating DNA replication (Blanchard et al., 2002; Burhans et al., 2002; Pelizon et al., 2002). These results show that multiple p53-independent, proteasomeand/or caspase-dependent pathways destroy the replication initiation protein cdc6, which is required for the assembly and/or maintenance of preRCs, at an early stage of apoptosis. The proteasome-dependent destruction of cdc6, which was induced by the DNAdamaging drugs, occurs upstream of or parallel to the action of caspases. Thus, the destruction of cdc6 may be a primordial programmed cell death response that uncouples DNA replication from the cell division cycle. In this study, we report that MSC can potentiate cell lethality induced by topo I poison SN-38. Because SN-38 treatment in combination with MSC resulted in increased chk2 phosphorylation at Thr68 and down-regulation of DNA replication-associated proteins cdc6, MCM2, and cdc25A, accompanied by the induction of preapoptotic DNA fragmentation, we hypothesize that the alterations of chk2 phosphorylation and cdc6 expression were associated with the increased response of A253 cells to SN-38 by MSC. Materials and Methods Reagents. SN-38 was kindly supplied by Pharmacia Corporation (Kalamazoo, MI). The compound was dissolved in dimethyl sulfoxide with final dilutions made in the growth medium. MSC was obtained from Sigma Chemical Co. (St. Louis, MO). Proteinase K and RNase A were obtained from Roche Diagnostics Corp. (Indianapolis, IN). L-Methioninase (METase) was purchased from Wako Chemical USA, Inc. (Richmond, VA). The following antibodies were used: anti-chk1 monoclonal antibody (G-4), anti-chk2 polyclonal antibody (H-300), anti-cdc6 monoclonal antibody (180.2), and anti-cdc25A monoclonal antibody (F-6), anti-cdc25C, anti-14-3-3 monoand polyclonal antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA); phospho-chk1 (Ser345) antibody, phospho-chk2 (Thr68, Thr387, Thr432, and Ser19) antibody, and cleaved caspase-3 (Asp175) (5A1) rabbit monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA); and anti-human MCM2 monoclonal antibody (MBL Co., LTD, Nagoya, Japan). Anti-tubulin monoclonal antibody (clone TUB 2.1; Sigma) was used to demonstrate equal protein loading. All MSC treatment schedules included METase at 0.01 units/ml. Tissue Culture. Human head and neck carcinoma cell lines A253 (43-HTB) were purchased from American Type Culture Collection (Manassas, VA) and maintained as a monolayer in RPMI 1640 medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA). The doubling time of the cells is 27 h, and all treatments were carried out using exponentially growing cell cultures. The cell lines were free from mycoplasma as tested with the mycoplasma plus PCR primer set (Stratagene, Cedar Creek, TX)