Vorinostat and Mithramycin A in combination therapy as an interesting strategy for the treatment of Sézary T lymphoma: a transcriptomic approach.

SAHA (vorinostat) is a histone deacetylase inhibitor approved by the USA Food and Drug Administration (FDA) for treating advanced refractory cutaneous T cell lymphomas. As SAHA alters the expression of many genes under control of the Sp1 transcription factor, we examined the effect of its association with the FDA-approved anticancer antibiotic Mithramycin A (MTR, plicamycin), a competitive inhibitor of Sp1 binding to DNA. Sézary syndrome (SS) cells, expanded ex vivo from peripheral blood mononuclear cells of 4 patients, were tested for their sensitivity to the drugs regarding cytotoxicity and differential responsive gene expression. Multivariate statistical methods were used to identify genes whose expression is altered by SAHA, MTR, and the synergist effect of the two drugs. MTR, like SAHA, induced the apoptosis of SS cells, while the two drugs in combination showed clear synergy or potentiation. Expression data stressed a likely important role of additive or synergistic epigenetic modifications in the combined effect of the two drugs, while direct inhibition of Sp1-dependent transcription seemed to have only limited impact. Ontological analysis of modified gene expression suggested that the two drugs, either independently or synergistically, counteracted many intertwined pro-survival pathways deregulated in SS cells, resistance of these tumors to intrinsic and extrinsic apoptosis, abnormal adhesion migration, and invasive properties, as well as immunosuppressive behavior. Our findings provide preliminary clues on the individual and combined effects of SAHA and MTR in SS cells and highlight a potential therapeutic interest of this novel pair of drugs for treatment of SS patients.

Natural killer cells and malignant haemopathies: a model for the interaction of cancer with innate immunity.

Despite recent progress in the therapeutic approach of malignant haemopathies, their prognoses remain frequently poor. Immunotherapy offers an alternative of great interest in this context but defect or abnormal expression of human leukocyte antigens (HLA), frequently observed in cancer cells, limits its efficiency. Natural killer (NK) cells, which are able to kill target cells in a HLA-independent way, represent a novel tool in the treatment of haematological malignancies. Abnormal NK cytolytic function is observed in all the haematological malignancies studied, such as acute leukaemia, myelodysplastic syndromes or chronic myeloid/lymphoid leukaemia. Several mechanisms are involved in the alterations of NK cytotoxicity: decreased expression of activating receptors, increased expression of inhibitory receptors or defective expression of NK ligands on target cells. Further studies are needed to identify how each type of haematological malignancy escapes from the innate immune response. Attempts to increase the expression of activating receptors, to counteract inhibitory receptors expression, or to increase NK cell cytotoxic capacities could overcome tumour escape from innate immunity. These therapies are based on monoclonal antibodies or culture of NK cells in presence of cytokines or dendritic cells. Moreover, many novel drugs used in haematological malignancies [tyrosine kinase inhibitors, IMIDs(®), proteasome inhibitors, demethylating agents, histone deacetylase inhibitors (HDACis), histamine dihydrochloride] display interesting immunomodulatory properties that affect NK cells. These data suggest that combined modalities associating cytotoxic drugs with innate immunity modulators may represent a major breakthrough in tumour eradication.

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes.

A radioiodinated alpha-scorpion toxin (toxin II from Androctonus australis Hector) (alpha ScTx) was used as a probe for EM autoradiography to study the distribution of voltage-dependent sodium channels (Na+ channel) on the postsynaptic side of the mouse neuromuscular junction. Silver grain distribution was analyzed by the cross-fire method to assess the relative Na+ channel density in each membrane domain measured by stereology. This analysis showed that the maximum Na+ channel density was located on the edge of the synaptic gutter, where it reached about twice the mean density in the postsynaptic fold membrane. Na+ channel densities have been calculated using ACh receptor (AChR) density in fold crests as reference. Sodium channel density on the edge of the synaptic gutter was estimated at about 5000/microns 2. Sodium channel distribution in the postsynaptic folds was compared to AChR distribution using density distribution analysis (Fertuck and Salpeter, 1976). The results confirmed that, as already observed by immunogold labeling (Flucher and Daniels, 1989), there are no Na+ channels on fold crests. Na+ channels are located in the rest of the fold membrane (bottom) and may be distributed according to two possible models. In the first, density would be uniformly high, although lower than on the gutter edge. In the second, density would decrease from the crest border, where the value was that of the gutter edge, to the fold end, where the value would be 50% lower. Based on the latter model, which was the "best-fit model," we propose that the postsynaptic membrane includes two domains. The first is the fold crest, which contains almost exclusively AChRs. This domain is devoted to reception-transduction of the chemical signal. The second includes both the fold bottom membrane and the perisynaptic membrane. Sodium channel density is highest along the crest border and decreases moving away. Its functions are the integration of postsynaptic potentials and generation-conduction of the muscle action potential.

Localization of voltage-sensitive sodium channels on the extrasynaptic membrane surface of mouse skeletal muscle by autoradiography of scorpion toxin binding sites.

Voltage-dependent sodium channels (Na+ channels) were localized by autoradiography on mouse skeletal muscle using both light and electron microscopy. 125I-scorpion toxins (ScTx) of both the alpha and beta type were used as probes. The specificity of labelling was verified by competitive inhibition with unlabelled toxin and by inhibition of alpha ScTx labelling in depolarizing conditions. Under light microscopy, the labelling of the myocyte surface appeared randomly distributed with both the alpha and beta toxins. No difference in the labelling density obtained with beta ScTx was observed between a 2 mm central segment of the fibre containing the endplate and an adjacent segment not containing the endplate. At the endplate, however, the beta ScTx binding site density was about seven fold higher at the edge of the synaptic primary clefts. This density decreased with distance from the synaptic cleft reaching the extrasynaptic value at 30-40 microns. An analysis of myocyte labelling using electron microscopy provided evidence for a specific, but very low labelling of the myocyte interior which can be attributed to the T-tubules. These results confirm a relatively high density of Na+ channels in a perijunctional zone about 50 microns in width, which could ensure the initial spread of the surface depolarization with a high safety factor, and a homogeneous distribution over the remaining surface with a low density evaluated at 5-10 per microns2. However, the very low labelling of T-tubules could be attributed mainly to a low density of tubular Na+ channels.