Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor κB Activation in HL60 Cells

Abstract

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor B (NFB) by this drug. Mitoxantrone activated NFB and stimulated IB␣ degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the ␤ isoform of topoisomerase II and express a truncated ␣ isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factorstimulated IB degradation. The poisoning of topoisomerase II with resultant DNA damage is therefore a critical signal for NFB activation.Agents that induce stress in cells, such as ionizing radiation, reactive oxygen species, and anti-neoplastic drugs, change the expression of many genes by affecting transcription factors, including AP1, NF-AT, NFB, 1 and Egr-1 (1-7). Such genes may encode proteins that determine the commitment of a cell to mitotic arrest, damage repair, proliferation, or even apoptosis. The regulation and role of the inducible transcription factor NFB has been studied intensely during recent years. NFB is present in diverse cell types, activated in response to diverse stimuli by complex signaling pathways involving several protein-protein interactions and phosphorylations (8 -10). Typically, NFB resides in the cytosol as a dimer composed of subunits belonging to the Rel family of proteins, the prototype comprising a p50 and p65/RelA subunit. p50/p65(RelA) heterodimers have a potent transcriptional activating potential, whereas p50 homodimers lack transactivation activity due to the absence of a transcriptional activation domain (9,11,12).NFB heterodimers are sequestered ...

Molecular alterations in apoptotic pathways after PKB/Akt-mediated chemoresistance in NCI H460 cells

“…These agents can exert their cytotoxic mode of action by inducing DNA damage irrespective of the cell cycle phase. Mitoxantrone and doxorubicin have been described to intercalate with DNA at high concentrations, but also to inhibit Topoisomerase II, thereby leading to persistant double-strand breaks of DNA (Bodley et al, 1989;Boland et al, 2000). Cisplatin has been shown to alkylate DNA and to lead to the activation of caspase 8 with subsequent initiation of a caspase cascade (Ferreira et al, 2000).…”

Signals from within: the DNA-damage-induced NF-κB response

“…In these conditions, the activation of NF-kB is strictly dependent on the presence of an intact nucleus and a drug-sensitive version of topoisomerase I or II enzyme, showing the intimate link between the formation of double-strand breaks (DSBs) and NF-kB activation. [25][26][27] Topoisomerases are enzymes that control the degree of DNA supercoiling. Type I topoisomerases (targeted by CPT) create transient single-strand breaks in DNA, necessary for the induction of transcription, while topoisomerase II enzymes (inhibited by etoposide-like and doxorubicin-like drugs) are more involved in DNA replication and cell division and therefore create transient DSBs.…”

“…31,34 Recent evidence demonstrates that ATM is also required for the activation of NF-kB in response to various genotoxic stress insults, expanding the known ATM-dependent network. 27,[37][38][39] Although the NF-kB response to IR is still controversialmany authors utilize IR doses that are high above clinically relevant doses (r2 Gy) 40 and clear differences in sensitivity to IR are observed between primary and transformed cell types 41 -different groups independently demonstrated that the NF-kB response to IR in ATM-deficient cells is abrogated, both in transformed cell types 37,38 as well as in primary mouse tissues. 38 Furthermore, also CPT and doxorubicin appear to induce NF-kB activation via an ATM-dependent pathway.…”

“…38 Furthermore, also CPT and doxorubicin appear to induce NF-kB activation via an ATM-dependent pathway. 27,39,42 Importantly, anthracyclinlike molecules that do not cause DNA breaks but activate NF-kB via unrelated pathways (presumably through the activation of PKC-d) do not need ATM. 42,43 Also, TNF and LPS activate NF-kB in an ATM-independent way, [37][38][39] demonstrating that ATM is particularly required in those NF-kB pathways activated by the presence of DNA lesions.…”

“…38 In line with this, several other groups failed to detect a role for DNA-PK in IR, mithoxanthrone, CPT or daunomycin-induced NF-kB activation. 27,38,39 Finally, ATR is a third member of PI3-like protein kinase family clearly implicated in the DNA damage response. 31 Knockouts of this gene are lethal and no human disease is known associated with mutations in Atr, unambiguously demonstrating the essential role of this kinase.…”