Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA ...double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.
Eva and Otto Pfister, Tom; Pfister, Kathy; Pfister, Peter
11/2019
eBook
Open access
Eva and Otto is a true story about German opposition and resistance to Hitler as revealed through the early lives of Eva Lewinski Pfister (1910–1991) and Otto Pfister (1900–1985). It is an intimate ...and epic account of two Germans—Eva born Jewish, Otto born Catholic—who worked with a little-known German political group that resisted and fought against Hitler in Germany before 1933 and then in exile in Paris before the German invasion of France in May 1940. After their improbable escapes from separate internment and imprisonment in Europe, Eva obtained refuge in America in October 1940 where she worked to rescue other endangered political refugees, including Otto, with the help of Eleanor Roosevelt. As revealed in recently declassified records, Eva and Otto later engaged in different secret assignments with the US Office of Strategic Services (OSS) in support of the Allied war effort. Despite their vastly different backgrounds, Eva and Otto gave each other hope and strength as they acted upon what they understood to be an ethical duty to help others threatened by fascism. The book provides a sobering insight into the personal risks and costs of a commitment to that duty. Their unusually beautiful writing—directed to each other in diaries and correspondence during two long periods of wartime separation—also reveals an unlikely and inspiring love story.
The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor ...prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.
Topoisomerase I (Top1) is a proven target for cancer therapeutics, and the level of Top1 in tumors has been used as a biomarker for chemotherapeutic efficacy. In this study, we report the development ...and validation of a two-site enzyme chemiluminescent immunoassay for Top1, which we used to measure Top1 levels in the NCI-60 cancer cell line panel. Top1 levels ranged from 0.9 to 9.8 ng/mL/microg protein extract in these cell lines. Levels varied both within and between cancer types but were generally highest in colon cancer and leukemia cell lines and lowest in central nervous system and renal cancer cell lines. Top1 mRNA levels in the NCI-60 cell lines were also measured by microarray; mRNA values generally showed a good correlation with protein levels (Pearson correlation = 0.8). When these expression levels were compared with the activity of the indenoisoquinoline class of Top1 inhibitors across the NCI-60 cell panel, low levels of Top1 were associated with increased resistance to these drugs. The results of our studies indicate that our Top1 assay can be used to quantify Top1 levels in untreated cells as well as cells treated with Top1 inhibitors and that the assay has the potential to be adapted for use in predicting clinical response to Top1-active antineoplastic agents.
Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been ...fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP), and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation enhanced Mn(II)-binding affinity, as evidenced by a 2.8-fold decrease in the K M of Mn(II) oxidation. Second, introducing a salt bridge through Lys179Arg mutation improved Glu35 and Glu181 coordination to Mn(II), decreasing K M 2.6-fold. Third, eliminating a steric clash that prevented Glu37 from orienting toward Mn(II) resulted in an 8.6-fold increase in k cat/K M, arising primarily from a 3.6-fold decrease in K M, with a K M value comparable to that of the native enzyme (0.28 mM vs 0.19 mM for Pleurotus eryngii MnP PS3). We further demonstrated that while the effects of Tyr36Phe and Lys179Arg mutations are additive, because involved in secondary-shell interactions to different ligands, other combinations of mutations were antagonistic because they act on different aspects of the Mn(II) coordination at the same residues. Finally, we showed that these MnCcP variants are functional models of MnP that mimic its activity in both Mn(II) oxidation and degradation of a phenolic lignin model compound and kraft lignin. In addition to achieving KM in a designed protein that is similar to the that of native enzyme, our results offer molecular insight into the role of noncovalent interactions around metal-binding sites for improving metal binding and overall activity; such insight can be applied to rationally enhance these properties in other metalloenzymes and their models.
Summary Background Hypertension can be controlled adequately with existing drugs such as angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Nevertheless, treatment success ...is often restricted by patients not adhering to treatment. Immunisation against angiotensin II could solve this problem. We investigated the safety and efficacy of CYT006-AngQb—a vaccine based on a virus-like particle—that targets angiotensin II to reduce ambulatory blood pressure. Methods In this multicentre, double-blind, randomised, placebo-controlled phase IIa trial, 72 patients with mild-to-moderate hypertension were randomly assigned with a computer-generated randomisation list to receive subcutaneous injections of either 100 μg CYT006-AngQb (n=24), 300 μg CYT006-AngQb (24), or placebo (24), at weeks 0, 4, and 12. 24-h ambulatory blood pressure was measured before treatment and at week 14. The primary outcomes were safety and tolerability. Analyses were done by intention to treat. This study is registered with ClinicalTrials.gov , number NCT00500786. Findings Two patients in the 100 μg group, three in the 300 μg group, and none in the placebo group discontinued study treatment. All patients were included in safety analyses; efficacy analyses did not include the five dropouts, for whom no data were available at week 14. Five serious adverse events were reported (two in the 100 μg group, two in the 300 μg group, and one in the placebo group); none were deemed to be treatment related. Most side-effects were mild, transient reactions at the injection site. Mild, transient influenza-like symptoms were seen in three patients in the 100 μg group, seven in the 300 μg group, and none in the placebo group. In the 300 μg group, there was a reduction from baseline in mean ambulatory daytime blood pressure at week 14 by −9·0/−4·0 mm Hg compared with placebo (p=0·015 for systolic and 0·064 for diastolic). The 300 μg dose reduced the early morning blood-pressure surge compared with placebo (change at 0800 h −25/−13 mm Hg; p<0·0001 for systolic, p=0·0035 for diastolic). Interpretation Immunisation with CYT006-AngQb was associated with no serious adverse events; most observed adverse events were consistent with local or systemic responses similar to those seen with other vaccines. The 300 μg dose reduced blood pressure in patients with mild-to-moderate hypertension during the daytime, especially in the early morning. Funding Cytos Biotechnology AG.
The location of the Trp radical and the catalytic function of the Fe(IV)O Trp191 •+ intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of ...the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr71 • and Tyr236 • is independent of the Fe(IV)O Trp191 •+ intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Å from the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol Murphy, E. J., et al. (2012) FEBS J. 279, 1632–1639. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr71 • as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248–255 and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665–11674. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557–8563. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.
In order to assess the risk of patients being exposed to an anti-AIDS medication contaminated with EMS we have performed in depth genotoxicity, general toxicity and DMPK investigations. The results ...of these studies are reported in the accompanying papers of this issue. Prior to starting our investigations we searched the literature for toxicity data on this well established mutagen with specific attention to dose–response relations in
in vivo genotoxicity studies, since, obviously,
in vivo data are pivotal for risk assessment. There are numerous published
in vivo genotoxicity studies on EMS, with generally 50
mg/kg – or higher – being the minimal dose used. The dose of 50
mg/kg induced effects in some, but not all studies, while the dose of 100
mg/kg was clearly positive in most studies, except for heritable mutations where a single dose of 100
mg/kg was not observed to induce measurable effects in post-meiotic stages and even the maximal dose of 250
mg/kg was negative in pre-meiotic stages of male germ cell development. For somatic cells, NOEL values could not be derived for any of the endpoints studied. Although a large number of genotoxicity studies are available, none of the studies was sufficiently detailed to allow unambiguous conclusions about the presence of a (practical) threshold. But in most cases the dose–responses show a sublinear relationship (i.e. the slope increases with dose) which indicates that the data would not be incompatible with a threshold dose–response relationship. This stands in contrast to data on ethylnitrosourea (ENU) which has been studied concommittantly with EMS in several
in vitro and
in vivo genotoxicity investigations. ENU generally appeared to induce genotoxic effects with linear dose relationships.
We also review the more limited data reported on teratogenicity and carcinogenicity of EMS. Induction of fetal malformations in mice appeared to have a NOEL of 100
mg/kg. Classical life-time carcinogenicity studies have not been performed with EMS. Induction of mammary, lung, kidney, brain, and liver tumors has been observed after various short term treatment regimes. In none of the published studies a no effect level was reported and no exposure data are available. Overall, the experimental data do not fully characterize the carcinogenic potential of EMS and are insufficient for a risk extrapolation to humans. Although the data on teratogenicity and carcinogenicity are insufficient for assessing dose-response relations it is generally accepted that the genotoxic property of EMS is at the base of the teratogenic and carcinogenic effects.
Based on a production accident Viracept (nelfinavir mesilate) tablets, an HIV protease inhibitor supplied by Roche outside the US, Canada and Japan was contaminated with relatively high levels of ...ethyl methanesulfonate (EMS) for at most 3 months in spring of 2007. On the basis of a wide variety of toxicological data including critical experiments for mutation induction under chronic exposure conditions and cross-species exposure scaling experiments to extrapolate to humans, we estimate the added risk of adverse effects (cancer, birth abnormalities, heritable defects) in any individual patient accidentally exposed to EMS via contaminated Viracept tablets in the context of this production accident as essentially zero.
Of critical important for this risk assessment are pivotal
in vivo genotoxicity studies (MNT, MutaMouse) providing evidence for ‘hockey-stick’, like dose–response relationships for the risk defining induction of gene mutations and chromosomal damage by EMS Gocke, E., Müller, L., Pfister, T., Buergin, H., 2009a. Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate. Toxicol. Lett.; Gocke, E., Müller, L., Pfister, T., 2009b. EMS in Viracept—initial (‘traditional’) assessment of risk to patients based on linear dose response relations. Toxicol. Lett.; Gocke, E., Müller, L., Ballantyne, M., Whitwell, J., Müller, L., 2009c. MNT and MutaMouse studies to definde the in vivo dose-response relations of the genotoxicity of EMS and ENU. Toxicol. Lett.. As outlined in Gocke and Wall Gocke, E., Wall, M., 2009. In vivo genotoxicity of EMS: Statistical assessment of the dose response curves. Toxicol. Lett., several statistical approaches are in support of a threshold model to best fit the data. The presence of clear no effect levels in bone marrow, liver and GI-tract tissue with several dose levels tested below the NOEL permits the calculation of safety factors with considerable confidence. In calculating the ratio of the NOEL dose in the animal studies (25
mg/kg/day) divided by the calculated maximal daily dose of the patients (1068
ppm EMS in 2.92
g Viracept tablets
=
2.75
mg EMS or 0.055
mg/kg for a 50
kg person) we derive a safety factor of 454 based on oral intake. Detailed absorption, distribution and metabolism studies in mice, rats and monkeys and with human surrogates
in vitro enable us to estimate the safety factors also for the calculated likely highest exposure (AUC and
C
max) of patients to EMS Lave, T., Birnböck, H., Götschi, A., Ramp, T., Pähler, A., 2009a.
In vivo and
in vitro characterization of ethyl methanesulfonate pharmacokinetics in animals and in human. Toxicol. Lett.; Lave, T., Paehler, A., Grimm, H.P., 2009b. Modelling of patient EMS exposure: translating pharmacokinetics of EMS in vitro and in animals into patients. Toxicol. Lett.. We calculate the total exposure (AUC) based safety factor to amount to at least 28. This lower value is due to the conservative prediction of a longer half-life of EMS in man versus mouse, rat and monkey. Based on the estimated human
C
max the safety factor for affected Viracept patients is calculated to be 370, as
C
max is mainly dependent on volume of distribution, which is not much different for EMS in different species. We consider that the total exposure based safety factor constitutes a minimal value since the considerations regarding evidence of error-free repair at sub-threshold concentrations argues in favor of using the highest EMS concentration (
C
max) rather than the AUC as basis for risk assessment. The ‘true value’ very likely lies somewhere between these two numbers as aspects such as repair enzyme availability and status of the cell cycle relative to the insult are important parameters that may not fully support safety factors based solely on
C
max estimates.
Potential adverse effects of EMS such as cancer, birth abnormalities and heritable effects are considered to be sequelae of its genotoxic activity. Hence, the thresholded dose–response relationships should also apply to these endpoints. We also provide a comprehensive discussion of the specific disease situation of the HIV infected target population and potential influences of co-medications on the susceptibilities and repair capacities of EMS induced DNA lesions.
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