Tony T. Huang, Ph.D.

Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair
Colnaghi, Luca; Jones, Mathew J K; Cotto-Rios, Xiomaris M; Schindler, Detlev; Hanenberg, Helmut; Huang, Tony T
2011 Feb 17;117(7):2247-2256, Blood
Fanconi anemia (FA) is a rare familial genome instability syndrome caused by mutations in FA genes that results in defective DNA crosslink repair. Activation of the FA pathway requires the FA core ubiquitin ligase complex-dependent monoubiquitination of 2 interacting FA proteins, FANCI and FANCD2. Although loss of either FANCI or FANCD2 is known to prevent monoubiquitination of its respective partner, it is unclear whether FANCI has any additional domains that may be important in promoting DNA repair, independent of its monoubiquitination. Here, we focus on an FA-I patient-derived FANCI mutant protein, R1299X (deletion of 30 residues from its C-terminus), to characterize important structural region(s) in FANCI that is required to activate the FA pathway. We show that, within this short 30 amino acid stretch contains 2 separable functional signatures, a nuclear localization signal and a putative EDGE motif, that is critical for the ability of FANCI to properly monoubiquitinate FANCD2 and promote DNA crosslink resistance. Our study enable us to conclude that, although proper nuclear localization of FANCI is crucial for robust FANCD2 monoubiquitination, the putative FANCI EDGE motif is important for DNA crosslink repair
— id: 124093, year: 2011, vol: 117, page: 2247, stat: Journal Article,

Insights into phosphorylation-dependent mechanisms regulating USP1 protein stability during the cell cycle
Cotto Rios XM; Jones MJ; Huang TT
2011 Dec 1;10(23):4009-4016, Cell cycle
Tight regulation of the cell cycle and DNA repair machinery is essential for maintaining genome stability. The APC/CCdh1 ubiquitin ligase complex is a key regulator of protein stability during the G 1 phase of the cell cycle. APC/CCdh1 regulates and promotes the degradation of proteins involved in both cell cycle regulation and DNA repair. In a recent study, we identified a novel APC/CCdh1 substrate, the ubiquitin protease USP1. USP1 is a critical regulator of both the Fanconi anemia (FA) and translesion synthesis (TLS) DNA repair pathways. Here, we provide additional mechanistic insights into the regulation of USP1 during the cell cycle. Specifically, we demonstrate that USP1 is phosphorylated in mitosis by cyclin-dependent kinases (Cdks), and that this phosphorylation event may prevent premature degradation of USP1 during normal cell cycle progression. Finally, we provide a unifying hypothesis integrating the role of G 1-specific proteolysis of USP1 with the regulation of the transcriptional repressors, Inhibitor of DNA-binding (ID) proteins
— id: 145997, year: 2011, vol: 10, page: 4009, stat: Journal Article,

APC/CCdh1-dependent proteolysis of USP1 regulates the response to UV-mediated DNA damage
Cotto-Rios, Xiomaris M; Jones, Mathew J K; Busino, Luca; Pagano, Michele; Huang, Tony T
2011 Jul 25;194(2):177-186, Journal of cell biology
Targeted protein destruction of critical cellular regulators during the G1 phase of the cell cycle is achieved by anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)), a multisubunit E3 ubiquitin ligase. Cells lacking Cdh1 have been shown to accumulate deoxyribonucleic acid (DNA) damage, suggesting that it may play a previously unrecognized role in maintaining genomic stability. The ubiquitin-specific protease 1 (USP1) is a known critical regulator of DNA repair and genomic stability. In this paper, we report that USP1 was degraded in G1 via APC/C(Cdh1). USP1 levels were kept low in G1 to provide a permissive condition for inducing proliferating cell nuclear antigen (PCNA) monoubiquitination in response to ultraviolet (UV) damage before DNA replication. Importantly, expression of a USP1 mutant that cannot be degraded via APC/C(Cdh1) inhibited PCNA monoubiquitination during G1, likely compromising the recruitment of trans-lesion synthesis polymerase to UV repair sites. Thus, we propose a role for APC/C(Cdh1) in modulating the status of PCNA monoubiquitination and UV DNA repair before S phase entry
— id: 135576, year: 2011, vol: 194, page: 177, stat: Journal Article,

Dysregulation of DNA polymerase kappa recruitment to replication forks results in genomic instability
Jones MJ; Colnaghi L; Huang TT
2011 Dec 13;:?-?, EMBO journal
Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non-pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polkappa to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polkappa to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin-binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polkappa-dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity
— id: 145996, year: 2011, vol: , page: ?, stat: Journal Article,

Nuclear export of the NF-kappaB inhibitor IkappaBalpha is required for proper B cell and secondary lymphoid tissue formation
Wuerzberger-Davis, Shelly M; Chen, Yuhong; Yang, David T; Kearns, Jeffrey D; Bates, Paul W; Lynch, Candace; Ladell, Nicholas C; Yu, Mei; Podd, Andrew; Zeng, Hu; Huang, Tony T; Wen, Renren; Hoffmann, Alexander; Wang, Demin; Miyamoto, Shigeki
2011 Feb 25;34(2):188-200, Immunity
The N-terminal nuclear export sequence (NES) of inhibitor of nuclear factor kappa B (NF-kappaB) alpha (IkappaBalpha) promotes NF-kappaB export from the cell nucleus to the cytoplasm, but the physiological role of this export regulation remains unknown. Here we report the derivation and analysis of genetically targeted mice harboring a germline mutation in IkappaBalpha NES. Mature B cells in the mutant mice displayed nuclear accumulation of inactive IkappaBalpha complexes containing a NF-kappaB family member, cRel, causing their spatial separation from the cytoplasmic IkappaB kinase. This resulted in severe reductions in constitutive and canonical NF-kappaB activities, synthesis of p100 and RelB NF-kappaB members, noncanonical NF-kappaB activity, NF-kappaB target gene induction, and proliferation and survival responses in B cells. Consequently, mice displayed defective B cell maturation, antibody production, and formation of secondary lymphoid organs and tissues. Thus, IkappaBalpha nuclear export is essential to maintain constitutive, canonical, and noncanonical NF-kappaB activation potentials in mature B cells in vivo
— id: 130274, year: 2011, vol: 34, page: 188, stat: Journal Article,

Calcium-dependent regulation of NEMO nuclear export in response to genotoxic stimuli
Berchtold, Craig M; Wu, Zhao-Hui; Huang, Tony T; Miyamoto, Shigeki
2007 Jan;27(2):497-509, Molecular & cellular biology
The mechanisms involved in activation of the transcription factor NF-kappaB by genotoxic agents are not well understood. Previously, we provided evidence that a regulatory subunit of the IkappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO)/IKKgamma, is a component of a nuclear signal that is generated after DNA damage to mediate NF-kappaB activation. Here, we found that etoposide (VP16) and camptothecin (CPT) induced increases in intracellular free calcium levels at 60 minutes after stimulation of CEM T leukemic cells. Inhibition of calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-kappaB activation by these agents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AM effects and promoted NF-kappaB activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16 treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclear exporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of a plausible calcium-dependent nuclear-export receptor calreticulin failed to prevent NF-kappaB activation by VP16. However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell line harboring a temperature sensitive mutant of RCC1 blocked NF-kappaB activation induced by genotoxic stimuli. Over-expression of Ran in this cell model showed that DNA damage stimuli induced formation of a complex between Ran and NEMO, suggesting that RCC1 regulated NF-kappaB activation through the modulation of RanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained by means of GST-pull down assays using GST fused to the Ran binding domain of RanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter these interactions suggesting that calcium is a necessary step beyond the formation of a Ran-GTP-NEMO complex in the nucleus. These results suggest that calcium has a unique role in genotoxic stress-induced NF-kappaB signaling by regulating nuclear export of NEMO subsequent to the formation of a nuclear export complex composed of Ran-GTP, NEMO and presumably an undefined nuclear export receptor
— id: 69204, year: 2007, vol: 27, page: 497, stat: Journal Article,

A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway
Cohn, Martin A; Kowal, Przemyslaw; Yang, Kailin; Haas, Wilhelm; Huang, Tony T; Gygi, Steven P; D'Andrea, Alan D
2007 Dec 14;28(5):786-797, Molecular cell
The deubiquitinating enzyme USP1 controls the cellular levels of the DNA damage response protein Ub-FANCD2, a key protein of the Fanconi anemia DNA repair pathway. Here we report the purification of a USP1 multisubunit protein complex from HeLa cells containing stoichiometric amounts of a WD40 repeat-containing protein, USP1 associated factor 1 (UAF1). In vitro reconstitution of USP1 deubiquitinating enzyme activity, using either ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) or purified monoubiquitinated FANCD2 protein as substrates, demonstrates that UAF1 functions as an activator of USP1. UAF1 binding increases the catalytic turnover (kcat) but does not increase the affinity of the USP1 enzyme for the substrate (KM). Moreover, we show that DNA damage results in an immediate shutoff of transcription of the USP1 gene, leading to a rapid decline in the USP1/UAF1 protein complex. Taken together, our results describe a mechanism of regulation of the deubiquitinating enzyme, USP1, and of DNA repair
— id: 130275, year: 2007, vol: 28, page: 786, stat: Journal Article,

FANCI is a second monoubiquitinated member of the Fanconi anemia pathway
Sims, Ashley E; Spiteri, Elizabeth; Sims, Robert J 3rd; Arita, Adriana G; Lach, Francis P; Landers, Thomas; Wurm, Melanie; Freund, Marcel; Neveling, Kornelia; Hanenberg, Helmut; Auerbach, Arleen D; Huang, Tony T
2007 Jun;14(6):564-567, Nature structural & molecular biology
Activation of the Fanconi anemia (FA) DNA damage-response pathway results in the monoubiquitination of FANCD2, which is regulated by the nuclear FA core ubiquitin ligase complex. A FANCD2 protein sequence-based homology search facilitated the discovery of FANCI, a second monoubiquitinated component of the FA pathway. Biallelic mutations in the gene coding for this protein were found in cells from four FA patients, including an FA-I reference cell line.
— id: 72707, year: 2007, vol: 14, page: 564, stat: Journal Article,

HAUSP hunting the FOX(O)
Huang, Tony T; D'Andrea, Alan D
2006 Oct;8(10):1043-1045, Nature cell biology
— id: 69205, year: 2006, vol: 8, page: 1043, stat: Journal Article,

Regulation of DNA repair by ubiquitylation
Huang, Tony T; D'Andrea, Alan D
2006 May;7(5):323-334, Nature reviews. Molecular cell biology
The process of ubiquitylation is best known for its role in targeting proteins for degradation by the proteasome. However, recent studies of DNA-repair and DNA-damage-response pathways have significantly broadened the scope of the role of ubiquitylation to include non-proteolytic functions of ubiquitin. These pathways involve the monoubiquitylation of key DNA-repair proteins that have regulatory functions in homologous recombination and translesion DNA synthesis, and involve the polyubiquitylation of nucleotide-excision-repair proteins
— id: 69206, year: 2006, vol: 7, page: 323, stat: Journal Article,

Regulation of monoubiquitinated PCNA by DUB autocleavage
Huang, Tony T; Nijman, Sebastian M B; Mirchandani, Kanchan D; Galardy, Paul J; Cohn, Martin A; Haas, Wilhelm; Gygi, Steven P; Ploegh, Hidde L; Bernards, Rene; D'Andrea, Alan D
2006 Apr;8(4):339-347, Nature cell biology
Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB
— id: 69207, year: 2006, vol: 8, page: 339, stat: Journal Article,

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway
Nijman, Sebastian M B; Huang, Tony T; Dirac, Annette M G; Brummelkamp, Thijn R; Kerkhoven, Ron M; D'Andrea, Alan D; Bernards, Rene
2005 Feb 4;17(3):331-339, Molecular cell
Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2
— id: 69208, year: 2005, vol: 17, page: 331, stat: Journal Article,

Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress
Huang, Tony T; Wuerzberger-Davis, Shelly M; Wu, Zhao-Hui; Miyamoto, Shigeki
2003 Nov 26;115(5):565-576, Cell
The transcription factor NF-kappaB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-kappaB signaling pathways is NEMO/IKKgamma, the regulatory subunit of the cytoplasmic IkappaB kinase (IKK) complex. While NF-kappaB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Here, we show that genotoxic stress causes nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-kappaB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets
— id: 69209, year: 2003, vol: 115, page: 565, stat: Journal Article,

The zinc finger domain of NEMO is selectively required for NF-kappa B activation by UV radiation and topoisomerase inhibitors
Huang, Tony T; Feinberg, Shelby L; Suryanarayanan, Sainath; Miyamoto, Shigeki
2002 Aug;22(16):5813-5825, Molecular & cellular biology
Exposure of mammalian cells to UV radiation was proposed to stimulate the transcription factor NF-kappa B by a unique mechanism. Typically, rapid and strong inducers of NF-kappa B, such as tumor necrosis factor alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS), lead to rapid phosphorylation and proteasomal degradation of its inhibitory protein, I kappa B alpha. In contrast, UV, a relatively slower and weaker inducer of NF-kappa B, was suggested not to require phosphorylation of I kappa B alpha for its targeted degradation by the proteasome. We now provide evidence to account for this peculiar degradation process of I kappa B alpha. The phospho-I kappa B alpha generated by UV is only detectable by expressing a Delta F-box mutant of the ubiquitin ligase beta-TrCP, which serves as a specific substrate trap for serine 32 and 36 phosphorylated I kappa B alpha. In agreement with this finding, we also find that the I kappa B kinase (IKK) phospho-acceptor sites on I kappa B alpha, core components of the IKK signalsome, and IKK catalytic activity are all required for UV signaling. Furthermore, deletion and point mutation analyses reveal that both the amino-terminal IKK-binding and the carboxy-terminal putative zinc finger domains of NEMO (IKK gamma) are critical for UV-induced NF-kappa B activation. Interestingly, the zinc finger domain is also required for NF-kappa B activation by two other slow and weak inducers, camptothecin and etoposide. In contrast, the zinc finger module is largely dispensable for NF-kappa B activation by the rapid and strong inducers LPS and TNF-alpha. Thus, we suggest that the zinc finger domain of NEMO likely represents a point of convergence for signaling pathways initiated by slow and weak NF-kappa B-activating conditions
— id: 69210, year: 2002, vol: 22, page: 5813, stat: Journal Article,

Postrepression activation of NF-kappaB requires the amino-terminal nuclear export signal specific to IkappaBalpha
Huang, T T; Miyamoto, S
2001 Jul;21(14):4737-4747, Molecular & cellular biology
One of the most prominent NF-kappaB target genes in mammalian cells is the gene encoding one of its inhibitor proteins, IkappaBalpha. The increased synthesis of IkappaBalpha leads to postinduction repression of nuclear NF-kappaB activity. However, it is unknown why IkappaBalpha, among multiple IkappaB family members, is involved in this process and what significance this feedback regulation has beyond terminating NF-kappaB activity. Herein, we report an important IkappaBalpha-specific function dictated by its amino-terminal nuclear export sequence (N-NES). The IkappaBalpha N-NES is necessary for the postinduction export of nuclear NF-kappaB, which is a critical event in reestablishing a permissive condition for NF-kappaB to be rapidly reactivated. We show that although IkappaBalpha and another IkappaB member, IkappaBbeta, can enter the nucleus and repress NF-kappaB DNA-binding activity during the postinduction phase, only IkappaBalpha allows the efficient export of nuclear NF-kappaB. Moreover, swapping the N-terminal region of IkappaBbeta for the corresponding IkappaBalpha sequence is sufficient for the IkappaB chimera protein to export NF-kappaB similarly to IkappaBalpha during the postinduction state. Our findings provide a mechanistic explanation of why IkappaBalpha but not other IkappaB members is crucial for postrepression activation of NF-kappaB. We propose that this IkappaBalpha-specific function is important for certain physiological and pathological conditions where NF-kappaB needs to be rapidly reactivated
— id: 130276, year: 2001, vol: 21, page: 4737, stat: Journal Article,

A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes
Huang, T T; Kudo, N; Yoshida, M; Miyamoto, S
2000 Feb 1;97(3):1014-1019, Proceedings of the National Academy of Sciences of the United States of America
Appropriate subcellular localization is crucial for regulation of NF-kappaB function. Herein, we show that latent NF-kappaB complexes can enter and exit the nucleus in preinduction states. The nuclear export inhibitor leptomycin B (LMB) sequestered NF-kappaB/IkappaBalpha complexes in the nucleus. Using deletion and site-directed mutagenesis, we identified a previously uncharacterized nuclear export sequence in residues 45-54 of IkappaBalpha that was required for cytoplasmic localization of inactive complexes. This nuclear export sequence also caused nuclear exclusion of heterologous proteins in a LMB-sensitive manner. Importantly, a LMB-insensitive CRM1 mutant (Crm1-K1) abolished LMB-induced nuclear accumulation of the inactive complexes. Moreover, a cell-permeable p50 NF-kappaB nuclear localization signal peptide also blocked these LMB effects. These results suggest that NF-kappaB/IkappaBalpha complexes shuttle between the cytoplasm and nucleus by a nuclear localization signal-dependent nuclear import and a CRM1-dependent nuclear export. The LMB-induced nuclear complexes could not bind DNA and were inaccessible to signaling events, because LMB inhibited NF-kappaB activation without affecting the subcellular localization of upstream kinases IKKbeta and NIK. Our findings indicate that the dominant nuclear export over nuclear import contributes to the largely cytoplasmic localization of the inactive complexes to achieve efficient NF-kappaB activation by extracellular signals
— id: 130279, year: 2000, vol: 97, page: 1014, stat: Journal Article,

NF-kappaB activation by camptothecin. A linkage between nuclear DNA damage and cytoplasmic signaling events
Huang, T T; Wuerzberger-Davis, S M; Seufzer, B J; Shumway, S D; Kurama, T; Boothman, D A; Miyamoto, S
2000 Mar 31;275(13):9501-9509, Journal of biological chemistry
Activation of the transcription factor NF-kappaB by extracellular signals involves its release from the inhibitor protein IkappaBalpha in the cytoplasm and subsequent nuclear translocation. NF-kappaB can also be activated by the anticancer agent camptothecin (CPT), which inhibits DNA topoisomerase (Topo) I activity and causes DNA double-strand breaks during DNA replication to induce S phase-dependent cytotoxicity. Here we show that CPT activates NF-kappaB by a mechanism that is dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. NF-kappaB activation by CPT is dramatically diminished in cytoplasts and in CEM/C2 cells expressing a mutant Topo I protein that fails to bind CPT. This response is intensified in S phase cell populations and is prevented by the DNA polymerase inhibitor aphidicolin. In addition, CPT activation of NF-kappaB involves degradation of cytoplasmic IkappaBalpha by the ubiquitin-proteasome pathway in a manner that depends on the IkappaB kinase complex. Finally, inhibition of NF-kappaB activation augments CPT-induced apoptosis. These findings elucidate the progression of signaling events that initiates in the nucleus with CPT-Topo I interaction and continues in the cytoplasm resulting in degradation of IkappaBalpha and nuclear translocation of NF-kappaB to attenuate the apoptotic response
— id: 130278, year: 2000, vol: 275, page: 9501, stat: Journal Article,

Cellular and molecular responses to topoisomerase I poisons. Exploiting synergy for improved radiotherapy
Miyamoto, S; Huang, T T; Wuerzberger-Davis, S; Bornmann, W G; Pink, J J; Tagliarino, C; Kinsella, T J; Boothman, D A
2000 ;922:274-292, Annals of the New York Academy of Sciences
The efficacy of topoisomerase (Topo) I-active drugs may be improved by better understanding the molecular and cellular responses of tumor compared to normal cells after genotoxic insults. Ionizing radiation (IR) + Topo I-active drugs (e.g., Topotecan) caused synergistic cell killing in various human cancer cells, even in cells from highly radioresistant tumors. Topo I poisons had to be added either during or immediately after IR. Synergy was caused by DNA lesion modification mechanisms as well as by concomitant stimulation of two pathways of cell death: necrosis (IR) + apoptosis (Topo I poisons). Cumulative data favor a mechanism of synergistic cell killing caused by altered DNA lesion modification and enhanced apoptosis. However, alterations in cell cycle regulation may also play a role in the synergy between these two agents in certain human cancers. We recently showed that NF-kappa B, a known anti-apoptotic factor, was activated in various cancer cells after poisoning Topo I using clinically active drugs. NF-kappa B activation was dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. Cytoplasmic signaling leading to NF-kappa B activation after Topo I poisons was diminished in cytoplasts (lacking nuclei) and in CEM/C2 cells that expressed a mutant Topo I protein that did not interact with Topo I-active drugs. NF-kappa B activation was intensified in S-phase and blocked by aphidicolin, suggesting that activation was a result of double-strand break formation due to Topo I poisoning and DNA replication. Dominant-negative I kappa B expression augmented Topo I poison-mediated apoptosis. Elucidation of molecular signal transduction pathways after Topo I drug-IR combinations may lead to improved radiotherapy by blocking anti-apoptotic NF-kappa B responses. Recent data also indicate that synergy caused by IR + Topo I poisons is different from radiosensitization by beta-lapachone (beta-lap), a 'reported' Topo I and II-alpha poison in vitro. In fact, beta-lap does not kill cells by poisoning either Topo I or II-alpha in vivo. Instead, the compound is 'activated' by an IR (damage)-inducible enzyme, NAD(P)H:quinone oxidoreductase (NQO1), a gene cloned as x-ray-inducible transcript #3, xip3. Unlike the lesion modification pathway induced by IR + Topo I drugs, beta-lap kills cells via NQO1 futile cycle metabolism. Downstream apoptosis caused by beta-lap appears to be noncaspase-mediated, involving calpain or a calpain-like protease. Thus, although Topo I poisons or beta-lap in combination with IR both synergistically kill cancer cells, the mechanisms are very different
— id: 130277, year: 2000, vol: 922, page: 274, stat: Journal Article,