Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2␣ is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2 is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2␣-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2␣ and not Top2 in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an ␣ isoform-specific manner. Heterozygous disruption of the human TOP2␣ gene confers increased NK314 resistance, whereas TOP2 homozygous knock-out cells display increased NK314 sensitivity, indicating that the ␣ isoform is the cellular target. We further show that the absence of Top2 does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2␣-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents. DNA topoisomerase II (Top2)2 is a ubiquitous nuclear enzyme that alters the topological structure of DNA and chromosomes through a transient DNA double-strand break (DSB) and subsequent religation of the DSB (1, 2). The enzyme has been implicated in many aspects of DNA metabolism, including DNA replication, repair, transcription, and chromosome condensation/segregation (1, 3). Top2 has been of considerable interest to human medicine, because it is an important target for cancer chemotherapy (4). Top2-targeting agents, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective and widely used anticancer drugs in cancer chemotherapy (5, 6). These agents are referred to as "Top2 poisons," because they convert the essential enzyme into a highly cytotoxic DNA-damaging agent through the formation of "cleavage complex" (also called "cleavable complex"), in which a Top2-linked DNA strand-passing intermediate is stabilized, allowing the generation of a DSB (7,8).Mammalian cells possess two genetically distinct Top2 isoforms (9, 10). Despite their similar structural features (ϳ70% identity at the amino acid level) and biological properties, the two isoforms are differentially regulated and play different roles in living cells. Top2␣ is most abundantly expressed in rapidly growing tissues and its expression is cell cycle-regulated...
Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. In this study, we found that the overexpression of the Bcl-2 family member Bax induced apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleaved the DEVD tetrapeptide. This apoptotic cell death was suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH 2 -DCB and zVAD-fmk. We also found that the Bax-induced apoptosis of COS-7 cells was suppressed by Bcl-x L and Bcl-2, though both Bcl-x L and Bcl-2 similarly prevented etoposide-induced apoptosis in COS-7 cells. In addition, Bcl-x L inhibited the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death but Bcl-2 did not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-x L to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells could be dierentially regulated. Our results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity.
Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that, similar to Bcl-2, Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, we examined the e ect of Pim-1 expression on c-Myc-mediated apoptosis of Rat-1 ®broblasts triggered by serum deprivation. Our results showed that, rather than inhibiting apoptosis, Pim-1 expression stimulated cMyc-mediated apoptosis in Rat-1 ®broblasts. Pim-1 stimulated c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a speci®c caspase inhibitor abolished the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant failed to stimulate c-Myc-mediated apoptosis, and Pim-1 expression alone in the absence of c-Myc overexpression did not induce apoptosis of serumdeprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway were required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 ®broblasts. Our results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) which regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation.
Antiestrogen agents are commonly used to treat patients with estrogen receptor (ER)-positive breast cancer. Tamoxifen has been the mainstay of endocrine treatment for patients with early and advanced breast cancer for many years. Following tamoxifen treatment failure, however, there are still limited options for subsequent hormonal therapy. We discovered a novel compound, NK150460, that inhibits 17β-estradiol (E2)-dependent transcription without affecting binding of E2 to ER. Against our expectations, NK150460 inhibited growth of not only most ER-positive, but also some ER-negative breast cancer cell lines, while never inhibiting growth of non-breast cancer cell lines. Cell-based screening using a random shRNA library, identified aryl hydrocarbon receptor nuclear translocator (ARNT) as a key gene involved in NK150460's antitumor mechanism. siRNAs against not only ARNT but also its counterpart aryl hydrocarbon receptor (AhR) and their target protein, CYP1A1, dramatically abrogated NK150460's growth-inhibitory activity. This suggests that the molecular cascade of AhR/ARNT plays an essential role in NK150460's antitumor mechanism. Expression of ERα was decreased by NK150460 treatment, and this was inhibited by an AhR antagonist. Unlike two other AhR agonists now undergoing clinical developmental stage, NK150460 did not induce histone H2AX phosphorylation or p53 expression, suggesting that it did not induce a DNA damage response in treated cells. Cell lines expressing epithelial markers were more sensitive to NK150460 than mesenchymal marker-expressing cells. These data indicate that NK150460 is a novel AhR agonist with selective antitumor activity against breast cancer cell lines, and its features differ from those of the other two AhR agonists.
Three types of transmembrane protein, IRE1/IRE1, PERK, and ATF6/ATF6 are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1 inhibitors. IRE1, IRE1, ATF6 and ATF6 are ubiquitously expressed in medaka. We found that IRE1/ATF6-double knockout is lethal, similarly to IRE1/IRE1-and ATF6/ATF6-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1 in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1 inhibitors in vivo.
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