RSK promotes G2/M transition through activating phosphorylation of Cdc25A and Cdc25B.

Activation of the mitogen-activated protein kinase (MAPK) cascade in mammalian cell lines positively regulates the G2/M transition. The molecular mechanism underlying this biological phenomenon remains poorly understood. Ribosomal S6 kinase (RSK) is a key downstream element of the MAPK cascade. Our previous studies established roles of RSK2 in Cdc25C activation during progesterone-induced meiotic maturation of Xenopus oocytes. In this study we demonstrate that both recombinant RSK and endogenous RSK in Xenopus egg extracts phosphorylate all three isoforms of human Cdc25 at a conserved motif near the catalytic domain. In human HEK293 and PC-3mm2 cell lines, RSK preferentially phosphorylates Cdc25A and Cdc25B in mitotic cells. Phosphorylation of the RSK sites in these Cdc25 isoforms increases their M-phase-inducing activities. Inhibition of RSK-mediated phosphorylation of Cdc25 inhibits G2/M transition. Moreover, RSK is likely to be more active in mitotic cells than in interphase cells, as evidenced by the phosphorylation status of T359/S363 in RSK. Together, these findings indicate that RSK promotes G2/M transition in mammalian cells through activating phosphorylation of Cdc25A and Cdc25B.

MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex.

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-X(L), ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Adenovirus 5 E1A enhances histone deacetylase inhibitors-induced apoptosis through Egr-1-mediated Bim upregulation.

Histone deacetylase inhibitors (HDACi) are potent anti-cancer agents for variety of cancer types. Suberoylanilide hydroxamic acid (SAHA) has been approved as a drug to treat cutaneous T cell lymphoma, and the combination of HDACi and other agents have been actively tested in many clinical trials. Adenovirus 5 early region 1A (E1A) has been shown to exhibit high tumor suppressor activity, and gene therapy using E1A has been tested in clinical trials. Here, we showed that proapoptotic activity of HDACi was robustly enhanced by E1A in multiple cancer cells, but not in normal cells. Moreover, we showed that combination of E1A gene therapy and SAHA showed high therapeutic efficacy with low toxicity in vivo ovarian and breast xenograft models. SAHA downregulated Bcl-XL and upregulated proapoptotic BH3-only protein Bim, whose expression was further enhanced by E1A in cancer cells. These alterations of Bcl-2 family proteins were critical for apoptosis induced by the combination in cancer cells. SAHA enhanced acetylation of histone H3 in Bim promoter region, while E1A upregulated Egr-1, which was directly involved in Bim transactivation. Together, our results provide not only a novel insight into the mechanisms underlying anti-tumor activity of E1A, but also a rationale for the combined HDACi and E1A gene therapy in future clinical trials.

Synthesis of N3-substituted thymidine analogues for measurement of cellular kinase activity.

N3-Substitued thymidine analogues that carry a carboranylalkyl moiety at the N3-position with various spacer lengths have been reported to be good substrates for thymidine kinase (TK1). As part of our continuing effort towards the development of new TK1 substrates for imaging tumor proliferative activity, we have synthesized a series of new N3-substituted analogues of thymidine that carry an aromatic ring with different spacer lengths. The overall yields for 6 and 7 were 13% and 39% in four steps and three steps, respectively, and those for 14, 16 and 18 were in the range of 13%-15% in six steps. The overall yield for 24 was 33% in three steps, and those for 25 and 26 were 64% and 58%, respectively, in one step. Most of these compounds have been tested for TK1 activity by enzymatic assay to identify a good substrate that can be radiolabeled for imaging. The phosphorylation rates of these compounds were 2%-6% compared with that of thymidine. The results from the in vitro enzymatic assays suggest that these N3-substituted thymidine analogues have some potential for imaging TK1 activity if radiolabeled with a suitable isotope.

Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway.

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

Molecular imaging of EGFR kinase activity in tumors with 124I-labeled small molecular tracer and positron emission tomography.

Positron emission tomography (PET) with epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide the means for noninvasive and repetitive imaging of heterogeneity of EGFR expression and signaling activity in tumors in individual patients before and during therapy with EGFR signaling inhibitors. We developed the synthesis and (124)I-radiolabeling of the (E)-But-2-enedioic acid [4-(3-[(124)I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-yl-propyl)-amide (morpholino-[(124)I]-IPQA), which selectively, irreversibly, and covalently binds the adenosine-triphosphate-binding site to the activated (phosphorylated) EGFR kinase, but not to the inactive EGFR kinase. The latter was demonstrated using in silico modeling with crystal structures of the wild type and different gain-of-function mutants of EGFR kinases. Also, this was demonstrated by selective radiolabeling of the EGFR kinase domain with morpholino-[(131)I]-IPQA in A431 human epidermoid carcinoma cells and Western blot autoradiography. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention postwashout of morpholino-[(131)I]-IPQA in A431 epidermoid carcinoma and in U87 human glioma cells genetically modified to express the EGFRvIII mutant receptor, but not in the wild-type U87MG glioma cells under serum-starved conditions. Using morpholino-[(124)I]-IPQA, we obtained noninvasive PET images of EGFR activity in A431 subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown from K562 human chronic myeloid leukemia cells in immunocompromised rats and mice. Based on these observations, we suggest that PET imaging with morpholino-[(124)I]-IPQA should allow for identification of tumors with high EGFR kinase signaling activity, including brain tumors expressing EGFRvIII mutants and nonsmall-cell lung cancer expressing gain-of-function EGFR kinase mutants. Because of significant hepatobiliary clearance and intestinal reuptake of the morpholino-[(124)I]-IPQA, additional [(124)I]-IPQA derivatives with improved water solubility may be required to optimize the pharmacokinetics of this class of molecular imaging agents.

Efficacy of dual-specific Bcr-Abl and Src-family kinase inhibitors in cells sensitive and resistant to imatinib mesylate.

Monotherapy of chronic myeloid leukemia (CML) with imatinib mesylate has been cast into shadow by the evolution of clinical resistance during therapy. Resistance to imatinib can arise by multiple mechanisms including amplification or mutation of Bcr-Abl, and continuity of imatinib therapy is probably a poor option for either of these patient groups. Recently, however, a structurally distinct new class of drugs, the pyrido[2,3-d]pyrimidines, has been described, and these compounds are predicted to make different molecular contacts in the Abl kinase domain. These drugs potently target both the Bcr-Abl and Src-family kinase activities, both of which are thought to be relevant to survival of the leukemic cell. We asked whether these drugs could selectively induce cell death in murine cell line models of CML cells sensitive and resistant to imatinib by different mechanisms. We show that whereas the pyrido[2,3-d] pyrimidines are indeed highly potent in suppressing proliferation of Bcr-Abl-overexpressing imatinib-resistant cells, they are almost completely ineffective against cells expressing the T315I mutant. This implies that despite structural differences from imatinib, these drugs are unlikely to be useful in patients expressing this mutant Bcr-Abl protein, but may be effective in cases where selection of cells overexpressing the oncoprotein leads to refractoriness to imatinib.

Fluorescent monitoring of kinase activity in real time: development of a robust fluorescence-based assay for Abl tyrosine kinase activity.

Fluorescent biosensors hold great promise for drug discovery. Using a solid-phase version of protein semi-synthesis, we incorporated two fluorophores at specific sites within a truncated version of the c-Crk-II protein. The resulting fluorescent protein biosensor permits the real-time monitoring of Abl kinase activity and provides a robust and rapid method for assaying Abl kinase inhibitors.

Imaging transcriptional regulation of p53-dependent genes with positron emission tomography in vivo.

A noninvasive method for molecular imaging of the activity of different signal transduction pathways and the expression of different genes in vivo would be of considerable value. It would aid in understanding the role specific genes and signal transduction pathways have in various diseases, and could elucidate temporal dynamics and regulation at different stages of disease and during various therapeutic interventions. We developed and assessed a method for monitoring the transcriptional activation of endogenous genes by positron-emission tomography (PET) imaging. The HSV1-tk/GFP (TKGFP) dual reporter gene was used to monitor transcriptional activation of p53-dependent genes. A retrovirus bearing the Cis-p53/TKGFP reporter system was constructed in which the TKGFP reporter gene was placed under control of an artificial cis-acting p53-specific enhancer. U87 glioma and SaOS-2 osteosarcoma cells were transduced with this retrovirus and used to establish xenografts in rats. We demonstrated that DNA damage-induced up-regulation of p53 transcriptional activity correlated with the expression of p53-dependent downstream genes, such as p21, in U87 (wild-type p53), but not in SaOS-2 osteosarcoma (p53 -/-) cells. We showed that PET, with [(124)I]FIAU (2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-[(124)I]iodouracil) and the Cis-p53TKGFP reporter system, is sufficiently sensitive to image the transcriptional regulation of genes in the p53 signal transduction pathway. These imaging results were confirmed by independent measurements of p53 activity and the expression levels of downstream genes (e.g., p21) by using conventional molecular-biological assays. PET imaging of p53 transcriptional activity in tumor xenografts by using the Cis-p53TKGFP reporter system may be useful in assessing novel therapeutic approaches.

Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction.

Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.

On the interactivity of complex synthesis and tumor pharmacology in the drug discovery process: total synthesis and comparative in vivo evaluations of the 15-aza epothilones.

The total syntheses of 12,13,15-desoxy-15(S)-aza-epothilone B (aza-dEpoB; dEpoB-lactam) and 12,13,15-desoxy-15(R)-aza-epothilone B (15-epi-aza-dEpoB; 15-epi-dEpoB-lactam) have been accomplished via a highly convergent strategy. We have also successfully oxidized 12,13,15-desoxy-15(S)-aza-epothilone B to aza-epothilone B (aza-EpoB; EpoB-lactam). Aza-epothilone B has been advanced to phase I clinical trials by the Bristol-Myers Squibb group. Our synthesis is efficient and was amenable to the production of significant quantities of these lactams. Using our fully synthetically derived lactams, in vitro and in vivo studies were conducted in comparison with advanced clinical candidates, 12,13-desoxyepothilone B and 12,13-desoxyepothilone F, also derived by total synthesis.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: a phase I trial.

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

Huanglian, A chinese herbal extract, inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer cells.

Huanglian is an herb that is widely used in China for the treatment of gastroenteritis. We elected to determine whether huanglian could inhibit tumor cell growth by modulating molecular events directly associated with the cell cycle. Huanglian inhibited tumor growth and colony formation of gastric, colon, and breast cancer cell lines in a time- and dose-dependent manner. Cell growth was completely inhibited after 3 days of continuous drug exposure to 10 microg/ml of herb. This degree of growth inhibition was significantly greater than that observed with berberine, the major constituent of the herb. The inhibition of cell growth by huanglian was associated with up to 8-fold suppression of cyclin B1 protein. This resulted in complete inhibition of cdc2 kinase activity and accumulation of cells in G(2). The mRNA expression of cyclin B1 was not changed after huanglian treatment. There was no change in the protein expression of cyclins A or E. Therefore, the effect of huanglian on inhibiting tumor growth seems to be mediated by the selective suppression of cyclin B1, which results in the inhibition of cdc2 kinase activity. Inhibition of cyclin dependent kinase (cdk) activity is emerging as an attractive target for cancer chemotherapy. Huanglian represents a class of agents that can inhibit tumor cell growth by directly suppressing the expression of a cyclin subunit that is critical for cell cycle progression. These results indicate that traditional Chinese herbs may represent a new source of agents designed for selective inhibition of cyclin dependent kinases in cancer therapy.

Structural mechanism for STI-571 inhibition of abelson tyrosine kinase.

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.

Co-administration of probenecid, an inhibitor of a cMOAT/MRP-like plasma membrane ATPase, greatly enhanced the efficacy of a new 10-deazaaminopterin against human solid tumors in vivo.

Earlier studies from this laboratory have shown that the uricosuric agent probenecid (PBCD) will inhibit the extrusion of folate analogues from tumor cells mediated by a plasma membrane ATPase resembling the canicular multispecific organic anion transporter/multidrug resistance-related protein (MRP) family of ATP binding cassette transporters. This inhibition of this outwardly directed membrane ATPase has been shown to have a favorable impact upon the cellular pharmacokinetics, cytotoxicity, and efficacy of methotrexate in vivo. In an extension of these earlier studies, which had focused only on murine ascites tumors, we now report that parental co-administration of PBCD will also enhance net intracellular accumulation in vitro and intracellular persistence in vivo of a new folate analogue, 10-propargyl-10-deazaaminopterin (PDX) in tumor cells. This resulted in marked enhancement of the efficacy of PDX against murine and human lung neoplasms and human prostate and mammary neoplasms growing as solid tumors in mice. As possible ATPases targeted by PBCD, all of these tumors expressed MRP-1, -4, and -7 genes, with expression of MRP-4 being greatest in each case. Four other MRP genes were expressed to a variable extent in some tumors but not others. The therapeutic enhancement of PDX by PBCD was manifested as tumor regression, where PDX alone was only growth inhibitory (A549 NSCL tumor), or as a substantial increase (3-4-fold) in overall regression and/or number of complete regressions (Lewis and LX-1 lung, PC-3 and TSU-PR1 prostate, and MX-1 mammary tumors) compared to PDX alone. Also, only in the case of PDX with PBCD, a significant number of mice transplanted with LX-1 or MX-1 tumors that experienced complete regression did not have regrowth of their tumor. In view of these results, clinical trials of this therapeutic modality appear to be warranted, especially in the case of new more efficacious folate analogues that are also permeants for this canicular multispecific organic anion transporter/MRP-like plasma membrane ATPase.

Immunization of ovarian cancer patients with a synthetic Lewis(y)-protein conjugate vaccine: a phase 1 trial.

As the initial step in developing carbohydrate-based vaccines for the treatment of ovarian cancer patients in an adjuvant setting, 25 patients were immunized with a Lewis(y) pentasaccharide (Le(y))-keyhole limpet hemocyanin (KLH)-conjugate vaccine together with the immunological adjuvant QS-21. Four different doses of the vaccine, containing 3, 10, 30, and 60 microg of carbohydrate were administered s.c. at 0, 1, 2, 3, 7, and 19 weeks to groups of 6 patients. Sera taken from the patients at regular intervals were assayed by ELISA for reactivity with naturally occurring forms of Le(y) (Le(y)-ceramide and Le(y) mucin) and by flow cytometry and a complement-dependent cytoxicity assay for reactivity with Le(y)-expressing tumor cells. The majority of the patients (16/24) produced anti-Le(y) antibodies as assessed by ELISA, and a proportion of these had strong anti-tumor cell reactivity as assessed by flow cytometry and complement-dependent cytotoxicity. One serum, analyzed in detail, was shown to react with glycolipids but not with glycoproteins or mucins expressed by ovarian cancer cell line OVCAR-3. The vaccine was well tolerated and no gastrointestinal, hematologic, renal, or hepatic toxicity related to the vaccine was observed. On the basis of this study, Le(y)-KLH should be a suitable component for a polyvalent vaccine under consideration for the therapy of epithelial cancers.

Polyclonal antibodies from patients immunized with a globo H-keyhole limpet hemocyanin vaccine: isolation, quantification, and characterization of immune responses by using totally synthetic immobilized tumor antigens.

We have previously reported on a carbohydrate-based vaccine program for immunotherapy in cancer patients. One such vaccine, based on the globo H antigen conjugated to the protein keyhole limpet hemocyanin (KLH), has been in clinical evaluation. Although this and other carbohydrate vaccines have been shown to induce antibody responses, there are currently no quantitative data on the antibody levels achieved in immunized patients by these or other anti-cancer vaccines. We report herein an efficient route to complex synthetic oligosaccharides attached to an affinity matrix for identifying and isolating antibodies elicited against such a carbohydrate-based vaccine in humans. Pre- and postvaccination profiles from serum samples of patients immunized with globo H-KLH were compared. All anti-globo H antibody activity was efficiently separated from other serum constituents. The isolated antibodies were readily quantified, and their specificities were analyzed. Since no comparable data were available on antibodies resulting from the vaccination of other cancer patients, we compared the observed levels with those quoted in studies with bacterial polysaccharide vaccines that had been quantified. Remarkably, cancer patients immunized with globo H-KLH produce anti-globo H antibody levels often exceeding those formed by immunization with bacterial polysaccharides. In addition, substantial quantities of both IgG and IgM antibodies were elicited, clearly indicating a class switch to IgG. Taken together, these analyses serve to clarify several aspects of the immune response to the vaccine and give several new insights to the carbohydrate-based vaccination strategy. Furthermore, antibodies so isolated could well have applications in clinical therapy.

Cellular and molecular responses to topoisomerase I poisons. Exploiting synergy for improved radiotherapy.

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.