Using digital RNA counting and flow cytometry to compare mRNA with protein expression in acute leukemias.

BACKGROUND: The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients. METHODS: mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated. RESULTS: Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL). CONCLUSIONS: The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.

Isolation, structural assignment and total synthesis of Barmumycin

Barmumycin was isolated from an extract of the marine actinomycete Streptomyces sp. BOSC-022A and found to be cytotoxic against various human tumor cell lines. Based on preliminary one- and two-dimensional 1H- and 13C-NMR spectra, the natural compound was initially assigned the structure of macrolactone-type compound 1, which was later prepared by two different routes. However, major spectroscopic differences between isolated barmumycin and 1 led to revision of the proposed structure as E-16. Based on synthesis of this new compound, and subsequent spectroscopic comparison of it to an authentic sample of barmumycin, the structure of the natural compound was indeed confirmed as that of E-16.

Study of the incorporation and the anti-tumour efficacy of radio-iododeoxyuridine according to the cellular cycle and in presence of synthesis inhibitor of thymidine

Résumé La iododeoxyuridine (IdUrd), une fois marqué au 123I ou au 125I, est un agent potentiel pour des thérapies par rayonnements Auger. Cependant, des limitations restreignent son incorporation dans l'ADN. Afin d'augmenter celle-ci, différents groupes ont étudié la fluorodeoxyuridine (FdUrd), qui favorise l'incorporation d'analogue de la thymidine, sans toutefois parvenir à une toxicité associé plus importante. Dans notre approche, 3 lignées cellulaires de glioblastomes humains et une lignée de cancer ovarien ont été utilisées. Nous avons observé, 16 à 24 h après un court pré-traitement à la FdUrd, un fort pourcentage de cellules s'accumulant en phase S. Plus qu'une accumulation, c'était une synchronisation des cellules, celles-ci restant capables d'incorporer la radio-IdIrd et repartant dans le cycle cellulaire. De plus, ces cellules accumulées après un pré-traitement à la FdUrd étaient plus radio-sensibles. Après le même intervalle de 16 à 24 h suivant la FdUrd, les 4 lignées cellulaires ont incorporé des taux plus élevés de radio-IdUrd que sans ce prétraitement. Une corrélation temporelle entre l'accumulation des cellules en phase S et la forte incorporation de radio-IdUrd a ainsi été révélée 16 à 24 h après pré-traitement à la FdUrd. Les expériences de traitement par rayonnements Auger sur les cellules accumulées en phase S ont montré une augmentation significative de l'efficacité thérapeutique de 125I-IdUrd comparé aux cellules non prétraitées à la FdUrd. Une première estimation a permis de déterminer que 100 désintégrations de 125I par cellules étant nécessaires afin d'atteindre l'efficacité thérapeutique. De plus, p53 semble jouer un rôle dans l'induction directe de mort cellulaire après des traitements par rayonnements Auger, comme indiqué par les mesures par FACS d'apoptose et de nécrose 24 et 48 h après le traitement. Concernant les expériences in vivo, nous avons observé une incorporation marquée de la radio-IdUrd dans l'ADN après un pré-traitement à la FdUrd dans un model de carcinomatose ovarienne péritonéale. Une augmentation encore plus importante a été observée après injection intra-tumorale dans des transplants sous-cutanés de glioblastomes sur des souris nues. Ces modèles pourraient être utilisés pour de plus amples études de diffusion de radio-IdUrd et de thérapie par rayonnement Auger. En conclusion, ce travail montre une première application réussie de la FdUrd afin d'accroître l'efficacité de la radio-IdUrd par traitements aux rayonnements Auger. La synchronisation des cellules en phase S combinée avec la forte incorporation de radio-IdUrd dans l'ADN différées après un pré-traitement à la FdUrd ont montré le gain thérapeutique attendu in vitro. De plus, des études in vivo sont tout indiquées après les observations encourageantes d'incorporation de radio-IdUrd dans les models de transplants sous-cutanés de glioblastomes et de tumeurs péritonéales ovariennes. Summary Iododeoxyuridine (IdUrd), labelled with 123I or 125I, could be a potential Auger radiation therapy agent. However, limitations restrict its DNA incorporation in proliferating cells. Therefore, fluorodeoxyuridine (FdUrd), which favours incorporation of thymidine analogues, has been studied by different groups in order to increase radio-IdUrd DNA incorporation, however therapeutic efficacy increase could not be reached. In our approach, 3 human glioblastoma cell lines with different p53 expression and one ovarian cancer line were pre-treated with various FdUrd conditions. We observed a high percentage of cells accumulating in early S phase 16 to 24 h after a short and non-toxic FdUrd pre-treatment. More than an accumulation, this was a synchronization, cells remaining able to incorporate radio-IdUrd and re-entering the cell cycle. Furthermore, the S phase accumulated cells post FdUrd pre-treatment were more radiosensitive. After the same delay of 16 to 24 h post FdUrd pre-treatment, the 4 cell lines were incorporating higher rates of radio-IdUrd compared with untreated cells. A time correlation between S phase accumulation and high radio-IdUrd incorporation was therefore revealed 16 to 24 h post FdUrd pre-treatment. Auger radiation treatment experiments performed on S phase enriched cells showed a significant increase of killing efficacy of 125I-IdUrd compared with cells not pre-treated with FdUrd. A first estimation indicates further that about 100 125I decays were required to reach killing in the targeted cells. Moreover, p53 might play a role on the direct induction of cell death pathways after Auger radiation treatments, as indicated by differential apoptosis and necrosis induction measured by FACS 24 and 48 h after treatment initiation. Concerning in vivo results, we observed a marked DNA incorporation increase of radio-IdUrd after FdUrd pre-treatment in peritoneal carcinomatosis in SCID mice. Even higher incorporation increase was observed after intra-tumoural injection of radio-IdUrd in subcutaneous glioblastoma transplants in nude mice. These tumour models might be further useful for diffusion of radio-IdUrd and Auger radiation therapy studies. In conclusion, these data show a first successful application of thymidine synthesis inhibition able to increase the efficacy of radio-IdUrd Auger radiation treatment. The S phase synchronization combined with a high percentage DNA incorporation of radio-IdUrd delayed post FdUrd pre-treatment provided the expected therapeutic gain in vitro. Further in vivo studies are indicated after the observations of encouraging radio-IdUrd uptake experiments in glioblastoma subcutaneous xenografts and in an ovarian peritoneal carcinomatosis model.

Defective nitric oxide synthesis: a link between metabolic insulin resistance, sympathetic overactivity and cardiovascular morbidity.

Epidemiological studies demonstrate an association between insulin resistance, hypertension and cardiovascular morbidity. In addition to its metabolic effects, insulin also has important cardiovascular actions. The sympathetic nervous system and the nitric oxide-l-arginine pathway have emerged as central players in the mediation of these actions. Over the past decade, the underlying mechanisms and the factors that may govern the interaction between insulin and these two major cardiovascular regulatory systems have been studied extensively in healthy people and insulin-resistant individuals. Here we summarize the current understanding and gaps in knowledge on these interactions. We propose that a genetic and/or acquired defect of nitric oxide synthesis could represent a central defect triggering many of the metabolic, vascular and sympathetic abnormalities characteristic of insulin-resistant states, all of which may predispose to cardiovascular disease.