Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes

Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated a novel strategy for treatment of MTC, combining “suicide” and interleukin-2 (IL-2) gene therapies. Tumors were produced in Wag/Rij rats by orthotopic injection of the rMTC 6–23 cell line, and/or derivatives expressing the herpes simplex virus 1 thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient “bystander” effect in vivo. Double labelling of rMTC cell membranes and apoptotic nuclei revealed that, as with the TK⁺ cells, some TK⁻ cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8⁺ and CD4⁺ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management in man. Received: 1 October 1998 / Accepted: 1 January 1999     

Distribution of calcitonin gene-related peptide and calcitonin-like immunoreactivity in trout

Radioimmunoassay and chromatography were used to study the occurrence of calcitonin gene-related peptide in various tissues of the rainbow trout, Salmo gairdnerii. The highest concentrations of the peptide were found in gill (1.68 +/- 0.09 ng/mg protein) and in intestine (1.06 +/- 0.4 ng/mg protein). Significant concentrations were also found in heart and stomach. The level in brain was very low. In trout, the plasma concentration accounted for 283 +/- 82 pg/ml. Chromatographic analysis of the calcitonin gene-related peptide (CGRP)-like immunoreactivity occurring in gills showed that two molecular forms cross-reacted with the anti-human CGRP antibody, one co-eluting with the synthetic human CGRP. In addition, calcitonin in fish is not confined to the ultimobranchial organ but is also present in organs as heart, intestine, kidney, spleen and stomach. The evidence of CGRP in fish emphasizes the role of this hormone in evolution and leads us to investigate its physiological role in this species.     

Chloride secretion by semicircular canal duct epithelium is stimulated via beta 2-adrenergic receptors

The ductalepithelium of the semicircular canal forms much of the boundary betweenthe K⁺-rich luminal fluid and the Na⁺-richabluminal fluid. We sought to determine whether the net ion fluxproducing the apical-to-basal short-circuit current(I_sc) in primary cultures was due to anionsecretion and/or cation absorption and under control of receptoragonists. Net fluxes of ²²Na, ⁸⁶Rb, and³⁶Cl demonstrated a basal-to-apical Clsecretion that was stimulated by isoproterenol. Isoproterenol andnorepinephrine increased I_sc with anEC₅₀ of 3 and 15 nM, respectively, and isoproterenolincreased tissue cAMP of native canals with an EC₅₀ of 5 nM. Agonists for adenosine, histamine, and vasopressin receptors had noeffect on I_sc. Isoproterenol stimulation ofI_sc and cAMP was inhibited by ICI-118551(IC₅₀ = 6 µM for I_sc) but notby CGP-20712A (1 µM) in primary cultures, and similar results werefound in native epithelium. I_sc was partially inhibited by basolateral Ba²⁺ (IC₅₀ = 0.27 mM) and ouabain, whereas responses to genistein, glibenclamide, andDIDS did not fully fit the profile for CFTR. Our findings show that thecanal epithelium contributes to endolymph homeostasis by secretion ofCl under ₂-adrenergic control with cAMP assecond messenger, a process that parallels the adrenergic control ofK⁺ secretion by vestibular dark cells. The current workpoints to one possible etiology of endolymphatic hydrops in Meniere'sdisease and may provide a basis for intervention.

     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of gene expression by antisense nucleic acids]

The use of antisense RNA or of antisense oligonucleotides for the specific control of viral or cellular genes expression has undergone rapid developments recently; their respective advantages and drawbacks will be discussed. Progresses in oligonucleotides chemistry have lead to the synthesis of analogs with improved pharmacological properties. Besides the antisense approach, which usually targets translation initiation or splicing sites, it is possible to interfere specifically with gene expression through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense approach). A major problem encountered in the use of synthetic oligonucleotides is their delivery to their nuclear or cytoplasmic targets after cell uptake by an endocytic pathway; our own work in this field will be discussed. Finally, we will describe the strategies followed by our group to improve the bioavailability of antisense oligonucleotides, as for instance conjugation to poly (L-lysine) or encapsidation in antibody-targeted liposomes.     

Expression of the calcitonin gene during migration of the Pacific salmon, Oncorhynchus gorbuscha

Plasma calcitonin levels increase during reproduction in female salmon. Whether these changes in circulating levels of the hormone are due to increased secretion and or increased biosynthesis is not established. We measured total and poly A+ RNAs and calcitonin content of ultimobranchial bodies of male and female pink salmon during the different stages of the reproductive cycle. Calcitonin mRNAs were analysed by hybridization of northern and dot blots with a specific probe for salmon calcitonin. Dot blot autoradiography indicated that female animals had higher levels of CT mRNA than males and these values were correlated with plasma calcitonin levels. In conclusion, sexual hormones still to be identified, are probably implicated in the expression of the calcitonin gene in females during the reproductive cycle.     

The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.     

Interaction of filipin with dimyristoylphosphatidylcholine membranes studied by 2H-NMR,circular dichroism,electronic absorption and fluorescence

Interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine (DMPC) membranes has been monitored by ²H-NMR, circular dichroism (CD), electronic absorption and fluorescence in the temperature range 10° to 60°C. Interaction appears to depend on whether filipin is added before or after membrane formation and also upon the temperature of the system.When filipin is added to preformed DMPC large unilamellar vesicles (LUV), the association constants, as determined by electronic absorption are 39×10³ M ^-1, 15×10³ M ^-1 and 0.6×10³ M ^-1 at 15°, 30° and 50°C, respectively. Under identical conditions, CD spectra of bound filipin exhibit features characteristic of an aggregation over the whole temperature range.When filipin is incorporated in membranes during their preparation, the ²H-NMR spectra of deuterated DMPC indicate that the drug has a slight disordering effect on the lipid matrix below the temperature, T _c ,of the gel-to-fluid phase transition and above T _c +11°C. Between these two temperature boundaries the system consists of two lipid regions of very different dynamic properties. One of the regions, which is attributed to a filipin-lipid complex, has the properties of gel-like lipids whereas the other has those of fluid-like lipids. The latter domain is however more ordered than the pure lipid at corresponding temperatures. CD spectra under the same conditions are found to be identical to spectra when the drug is added to preformed membranes, only in the region T _c to T _c +11°C.Filipin induced carboxyfluorescein release from DMPC-LUV is found to be complete when the filipin-to-lipid ratio is near 1, for temperatures below T _c +11°C.Results are compared to previous data on amphotericin B and provide evidence that the gel-like structure of phospholipid and membrane permeation may be induced by filipin even in the absence of cholesterol.Abbreviations NMR nuclear magnetic resonance - CD circular dichroism - DMPC dimyristoylphosphatidylcholine - EPA egg phosphatidic acid - LUV large unilamelar vesicles - SPC soybean phosphatidylcholine - DMSO dimethylsulfoxide - CF carboxyfluorescein