Inhibitory effects of marine-derived DNA-binding anti-tumour tetrahydroisoquinolines on the Fanconi anaemia pathway.

BACKGROUND AND PURPOSE: We have previously shown that cells with a defective Fanconi anaemia (FA) pathway are hypersensitive to trabectedin, a DNA-binding anti-cancer tetrahydroisoquinoline (DBAT) whose adducts functionally mimic a DNA inter-strand cross link (ICL). Here we expand these observations to new DBATs and investigate whether our findings in primary untransformed cells can be reproduced in human cancer cells. EXPERIMENTAL APPROACH: Initially, the sensitivity of transformed and untransformed cells, deficient or not in one component of the FA pathway, to mitomycin C (MMC) and three DBATs, trabectedin, Zalypsis and PM01183, was assessed. Then, the functional interaction of these drugs with the FA pathway was comparatively investigated. KEY RESULTS: While untransformed FA-deficient haematopoietic cells were hypersensitive to both MMC and DBATs, the response of FA-deficient squamous cell carcinoma (SCC) cells to DBATs was similar to that of their respective FA-competent counterparts, even though these FA-deficient SCC cells were hypersensitive to MMC. Furthermore, while MMC always activated the FA pathway, the DBATs inhibited the FA pathway in the cancer cell lines tested and this enhanced their response to MMC. CONCLUSIONS AND IMPLICATIONS: Our data show that although DBATs functionally interact with DNA as do agents that generate classical ICL, these drugs should be considered as FA pathway inhibitors rather than activators. Moreover, this effect was most significant in a variety of cancer cells. These inhibitory effects of DBATs on the FA pathway could be exploited clinically with the aim of 'fanconizing' cancer cells in order to make them more sensitive to other anti-tumour drugs.

Development of an in vitro model for the simultaneous study of the efficacy and hematotoxicity of antileukemic compounds.

Hematopoietic system displays a wide spectrum of cell populations hierarchically organized in the bone marrow. Homeostasis in this system requires equilibrium between the self-renewal of the stem cells and their capacity of differentiation. Any failure on this equilibrium could lead to fatal consequences, such as the development of leukemia. Due to its rapid rate of renewal, hematopoietic tissue is a major target for antitumoral compounds and often becomes a dose limiting factor in the development of antineoplastics. Our aim was to develop an in vitro model for predicting the efficacy of antitumoral compounds on leukemic cells and their toxic effects on the healthy hematopoietic cells. The mouse myelomonocytic leukemia WEHI-3b was transduced with a lentiviral vector for expressing the green fluorescence protein. Mixed semisolid clonogenic cultures of transduced WEHI-3b and murine bone marrow cells were exposed to five pharmaceuticals: daunorubicin (positive control), atropine sulphate (negative control) and three in different stages of clinical development (trabectedin, Zalypsis(®) and PM01183). Colonies of leukemic cells were distinguishable from healthy CFU-GM under fluorescence microscope. The sensitivity of leukemic cells to daunorubicin, trabectedin, Zalypsis(®) and PM01183 was higher compared to healthy cells. The effect of a non-antitumoral compound, atropine sulphate, was the same on both populations. Our results show that this in vitro model is a valuable tool for studying the effect of antitumoral compounds in both tumoral and normal hematopoietic cells under the same toxic microenvironment and could safe time and facilitate the reduction of the number of animals used in preclinical development of pharmaceuticals.

Prevalidation of the rat CFU-GM assay for in vitro toxicology applications.

In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies - however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. In previous studies, the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood) and for predicting the in vivo human maximal tolerated dose (MTD) by adjusting in vivo data on mouse toxicity. Recently, a Colony Forming Unit-Megakaryocyte (CFU-MK) assay has also been prevalidated for testing drug toxicity toward megakaryocytes. The rat CFU-GM assay has been used by many researchers for its ability to evaluate in vitro haematotoxicity. Although it is not yet available, a standardised procedure for data comparison could be very important, since the rat is the most widely-used species for the in vivo testing of toxicants. This report presents the results of the prevalidation study developed to analyse the intra-laboratory and inter-laboratory variability of a standardised operating procedure for this assay and its performance for the in vitro determination of the inhibitory concentration (IC) values of drugs on rat myeloid progenitors (CFU-GM). The results demonstrate that the CFU-GM assay can be performed with cryopreserved rat bone-marrow cells (rBMC). The assay represents a useful tool for evaluating the toxicity of a compound, in terms of both relative toxicity (when different molecules are compared) and the prediction of the degree of in vivo toxicity. The use of this assay could greatly reduce the number of rats used in experimental procedures, and could also contribute to the accumulation of more toxicity data on compounds to be registered according to the criteria established by the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) programme.

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.

Phosphofructo-1-kinase deficiency leads to a severe cardiac and hematological disorder in addition to skeletal muscle glycogenosis.

Mutations in the gene for muscle phosphofructo-1-kinase (PFKM), a key regulatory enzyme of glycolysis, cause Type VII glycogen storage disease (GSDVII). Clinical manifestations of the disease span from the severe infantile form, leading to death during childhood, to the classical form, which presents mainly with exercise intolerance. PFKM deficiency is considered as a skeletal muscle glycogenosis, but the relative contribution of altered glucose metabolism in other tissues to the pathogenesis of the disease is not fully understood. To elucidate this issue, we have generated mice deficient for PFKM (Pfkm(-/-)). Here, we show that Pfkm(-/-) mice had high lethality around weaning and reduced lifespan, because of the metabolic alterations. In skeletal muscle, including respiratory muscles, the lack of PFK activity blocked glycolysis and resulted in considerable glycogen storage and low ATP content. Although erythrocytes of Pfkm(-/-) mice preserved 50% of PFK activity, they showed strong reduction of 2,3-biphosphoglycerate concentrations and hemolysis, which was associated with compensatory reticulocytosis and splenomegaly. As a consequence of these haematological alterations, and of reduced PFK activity in the heart, Pfkm(-/-) mice developed cardiac hypertrophy with age. Taken together, these alterations resulted in muscle hypoxia and hypervascularization, impaired oxidative metabolism, fiber necrosis, and exercise intolerance. These results indicate that, in GSDVII, marked alterations in muscle bioenergetics and erythrocyte metabolism interact to produce a complex systemic disorder. Therefore, GSDVII is not simply a muscle glycogenosis, and Pfkm(-/-) mice constitute a unique model of GSDVII which may be useful for the design and assessment of new therapies.

Yohimbine prevents the effect of morphine on the redox status of neuroblastomaxglioma NG108-15 cells.

The alpha(2)-adrenoceptor antagonist yohimbine is known to interact with the effects of opioid receptor agonists in vivo, and thus could modulate the action of morphine-like analgesics. The focus of the present work was to further study these interactions in a cell culture endowed with opioid and alpha(2)-adrenoceptors in order to know if they could happen at the cellular level. In a first step, incubation with morphine (10microM) or the delta opioid agonist DPDPE (1microM) for 6h was shown to decrease the reduction of (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) by NG108-15 neuroblastomaxglioma hybrid cells in a naloxone-sensitive manner, thus showing that the opioids affect the redox status of the cells in a delta receptor-mediated way. Further experiments with 2-24h incubation periods were subsequently performed with morphine 0.1microM, 10microM and 1mM and several tests to confirm the effects on metabolism (MTT, Alamar Blue tests) to examine the potential toxic consequences (neutral red test, trypan blue exclusion assay, LDH test, caspase 3/7 activity) and to study the potential effect of yohimbine on morphine toxicity. These studies confirmed that incubation with morphine (0.1microM and 10microM) affected to a similar extent the redox status of the cells, an effect that did not translated into significant cell death and was transient since completely disappeared after 24h of incubation. Morphine 1mM was much more toxic than the lower concentrations. Yohimbine effectively prevented the effects of the lower concentrations of morphine when added to the incubation medium at 10microM, a concentration devoid of significant toxicity. It seems that the exposure to pharmacologically relevant concentrations of morphine gives rise to short-term metabolic alterations of NG108-15 cells mediated by delta receptors and also sensitive to alpha(2)-adrenoceptor blockade; therefore, the interactions previously described in vivo between opioid and alpha(2)-adrenoceptor ligands do not necessarily require the presence of functional neuronal networks and they could happen at the cellular level.

In vitro sensitivity of granulo-monocytic progenitors as a new toxicological cell system and endpoint in the ACuteTox Project.

The ACuteTox Project (part of the EU 6th Framework Programme) was started up in January 2005. The aim of this project is to develop a simple and robust in vitro strategy for prediction of human acute systemic toxicity, which could replace animal tests used for regulatory purposes. Our group is responsible for the characterization of the effect of the reference chemicals on the hematopoietic tissue. CFU-GM assay based on the culture of human mononuclear cord blood cells has been used to characterize the effects of the selected compounds on the myeloid progenitors. Previous results have shown the relevance of the CFU-GM assay for the prediction of human acute neutropenia after treatment of antitumoral compounds, and this assay has been recently approved by the ECVAM's Scientific Advisory Committee. Among the compounds included in the study there were pharmaceuticals, environmental pollutants and industrial chemicals. Eleven out of 55 chemicals did not show any cytotoxic effect at the maximum concentration tested. The correlation coefficients of CFU-GM IC50, IC70 and IC90 values with human LC50 values (50% lethal concentration calculated from time-related sublethal and lethal human blood concentrations) were 0.4965, 0.5106 and 0.5142 respectively. Although this correlation is not improve respect to classical in vitro basal cytotoxicity tests such as 3T3 Neutral Red Uptake, chemicals which deviate substantially in the correlation with these assays (colchicine, digoxin, 5-Fluorouracil and thallium sulfate) fitted very well in the linear regression analysis of the CFU-GM progenitors. The results shown in the present study indicate that the sensitivity of CFU-GM progenitors correlates better than the sensitivity of HL-60 cells with human LC50 values and could help to refine the predictability for human acute systemic toxicity when a given chemical may affect to the hematopoietic myeloid system.

Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin.

Trabectedin (Yondelis; ET-743) is a potent anticancer drug that binds to DNA by forming a covalent bond with a guanine in one strand and one or more hydrogen bonds with the opposite strand. Using a fluorescence-based melting assay, we show that one single trabectedin-DNA adduct increases the thermal stability of the double helix by >20 degrees C. As deduced from the analysis of phosphorylated H2AX and Rad51 foci, we observed that clinically relevant doses of trabectedin induce the formation of DNA double-strand breaks in human cells and activate homologous recombination repair in a manner similar to that evoked by the DNA interstrand cross-linking agent mitomycin C (MMC). Because one important characteristic of this drug is its marked cytotoxicity on cells lacking a functional Fanconi anemia (FA) pathway, we compared the response of different subtypes of FA cells to MMC and trabectedin. Our data clearly show that human cells with mutations in FANCA, FANCC, FANCF, FANCG, or FANCD1 genes are highly sensitive to both MMC and trabectedin. However, in marked contrast to MMC, trabectedin does not induce any significant accumulation of FA cells in G2-M. The critical relevance of FA proteins in the response of human cells to trabectedin reported herein, together with observations showing the role of the FA pathway in cancer suppression, strongly suggest that screening for mutations in FA genes may facilitate the identification of tumors displaying enhanced sensitivity to this novel anticancer drug.

Use of CFU-GM assay for prediction of human maximum tolerated dose of a new antitumoral drug: Yondelis (ET-743).

Acute cytotoxic exposure causes decreases in bone marrow progenitors that precedes the neutrophil nadir. Experiments in animal models reveal a relationship between the reduction in granulocyte-macrophage progenitors (CFU-GM) and the decrease in absolute neutrophil count [Toxicol. Pathol. 21 (1993) 241]. Recently, the prevalidation of a model for predicting acute neutropenia by the CFU-GM assay has been reported [Toxicol. In Vitro 15 (2001) 729]. The model was based on prediction of human MTD by adjusting the animal-derived MTD for the differential sensitivity between CFU-GM from animal species and humans. In this study, this model has been applied on a new antitumoral drug, Yondelis (Ecteinascidin; ET-743). Preclinical studies showed that hematotoxicity was the main side effect in mice, being the MTD of 600 microg/m2 [Drugs Future 21 (1996) 1155]. The sensitivity of myeloid progenitors was higher in mice than in humans, with IC90 values of 0.69+/-0.22 nM and 1.31+/-0.21 nM for murine and human CFU-GMs respectively. This study predicts a human MTD of 1145 microg/m2. The reported human MTD of ET-743 given as a 24-h continuous infusion every 3 weeks is 1800 microg/m2 [J. Clin. Oncol. 19 (2001) 1256]. Since our predicted MTD is within fourfold of the actual MTD (the interspecies variation in tolerated dose due to differences in clearance rates, metabolism pathways and infusion rate) the result confirms the profit of the prediction model.

In vitro toxicity of three new antitumoral drugs (trabectedin, aplidin, and kahalalide F) on hematopoietic progenitors and stem cells.

OBJECTIVE: In addition to neutropenias and/or thrombocytopenias as a short-term effect, antineoplastics also can produce long-term effects as a consequence of damage to the hematopoietic stem cells. The aim of the present study was to evaluate the toxicity of three marine-derived antineoplastics on murine hematopoietic stem cells. These antitumoral compounds currently are being evaluated in patients in phase II (aplidin and kahalalide F) and phase II/III (trabectedin) clinical trials. MATERIALS AND METHODS: Long-term competitive repopulating assays were performed in mice to analyze toxic effects on the hematopoietic stem cells responsible for the multipotential long-term repopulation of hematopoiesis. Furthermore, granulocytic and T- and B-lymphoid lineages were studied, as well as myeloid (CFU-GM) and megakaryocytic (CFU-Meg) progenitors. RESULTS: When cells were treated in vitro for 24 hours with CFU-GM IC(50) dose of trabectedin (9.59+/-4.96 nM), no significant effects were observed in the stem cells. The dose of trabectedin that produced 90% of inhibition in CFU-GM (IC(90): 23.71+/-1.27 nM) only inhibited 45% survival of stem cells. Doses of aplidin that produced reductions of 50% (56.9+/-13.32 nM) or 90% (195.88+/-21.39 nM) in myeloid progenitors did not show any effect on hematopoietic stem cells. Kahalalide F did not show any toxic effect in either short-term or long-term repopulating cells up to 10 microM. CONCLUSIONS: Our data show that the hematopoietic stem cells effects of antitumoral drugs can be properly characterized by the murine competitive repopulating assays. Our results suggest that long-term myelosuppression as a consequence of trabectedin, aplidin, or kahalalide F treatment would not be expected.

In vitro tests for haematotoxicity: prediction of drug-induced myelosuppression by the CFU-GM assay.

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.