Role of near-infrared fluorescence imaging in the resection of metastatic lymph nodes in an optimized orthotopic animal model of HNSCC.

OBJECTIVES: To study the role of near-infrared fluorescence imaging in the detection and resection of metastatic cervical lymph nodes in head and neck cancer. MATERIALS AND METHODS: CAL33 head and neck cancer cells of human origin were implanted in the oral cavity of nude mice. The mice were followed up after tumor resection to detect the development of lymph node metastases. A specific fluorescent tracer for αvß3 integrin expressed by CAL33 cells was injected intravenously in the surviving mice between the second and the fourth month following tumor resection. A near-infrared fluorescence-imaging camera was used to detect tracer uptake in metastatic cervical lymph nodes, to guide of lymph-node resection for histological analysis. RESULTS: Lymph node metastases were observed in 42.8% of surviving mice between the second and the fourth month following orthotopic tumor resection. Near-infrared fluorescence imaging provided real-time intraoperative detection of clinical and subclinical lymph node metastases. These results were confirmed histologically. CONCLUSION: Near infrared fluorescence imaging provides real-time contrast between normal and malignant tissue, allowing intraoperative detection of metastatic lymph nodes. This preclinical stage is essential before testing the technique in humans.

Side-effects of a systemic injection of linear polyethylenimine-DNA complexes.

BACKGROUND: Systemic administration of linear polyethylenimine-DNA complexes (L-PEI/DNA) results in transient expression of the transgene in the lung. This study analyzes the side-effects associated with L-PEI-mediated transfection. METHODS: Mice weighing from 16 to 25 g received increasing amounts of L-PEI/DNA intravenously. Gene expression was evaluated using luciferase as a reporter gene. Toxicity was evaluated by monitoring the appearance of shock after injection, the survival of the animals, and the microscopic damage in the tissues. Adherence of blood cells and endothelium activation were observed after CD11-b and von Willebrand immunostaining. Anti-aggregant treatments were used in order to prevent the formation of thrombi. RESULTS: Increasing the quantity of L-PEI/DNA resulted in a marked augmentation of the luciferase activity in the lung, but was associated with liver necrosis and death. Lethality was reached at lower doses in older mice, suggesting an age influence. Transfection was associated with activation of the lung endothelium and increased adhesion of small aggregates containing platelets and CD11-b-positive cells, without the appearance of large thrombi and of lung injury. Anti-aggregant treatments (aspirin, EDTA, heparin or clopidogrel) decreased the L-PEI-mediated transfection, supporting the hypothesis that platelets participate in the blocking of DNA complexes in the lung capillaries. CONCLUSION: This study demonstrates that L-PEI/DNA activates the lung endothelium and forms small aggregates, a side-effect linked to the transfection efficiency.

Caffeine sensitizes human H358 cell line to p53-mediated apoptosis by inducing mitochondrial translocation and conformational change of BAX protein.

The mechanisms involved in p53-mediated cell death remain controversial. In the present study, we investigated this cell death pathway by stably transfecting the p53-null H358 cell line with a tetracycline-dependent wild type p53-expressing vector. Restoration of p53 triggered a G(2)/M cell cycle arrest and enhanced BAX protein expression, without inducing apoptosis or potentiating the cytotoxic effect of etoposide, vincristine, and cis-platinum. Accordingly, overexpression of BAX in H358 cells, through stable transfection of a tetracycline-regulated expression vector, did not induce cell death. Interestingly, the methylxanthine caffeine (4 mm) promoted the translocation of BAX from the cytosol to the mitochondria. In the setting of an overexpression of BAX, caffeine induced a conformational change of the protein and apoptosis. The consequences of caffeine were independent of its cell cycle-related activities. All together, caffeine synergizes with p53 for inducing cell death through a cell cycle-independent mechanism, involving mitochondrial translocation and conformational change of BAX protein.

Cell cycle arrest is sufficient for p53-mediated tumor regression.

p53 gene therapy can induce tumor regression, but the low efficacy of in vivo gene transfer has greatly hampered the mechanistic analysis of this antitumoral activity. We therefore used a p53-null human NSCLC cell line in which we reintroduced the wild-type p53 gene under control of a tetracycline-dependent promoter. P53 induction provokes cell cycle arrest in G0/G1 and G2/M phase, an up-regulation of p21, a down-regulation of cyclin B1 and appearance of senescence features without down-regulation of human telomerase reverse transcriptase. No detectable morphological changes of apoptosis nor procaspase-3 activation are observed. In subcutaneous tumors grafted in nude mice, the induction of p53 expression leads to a complete and longlasting tumor regression in 28 days which is associated with cell cycle arrest, but not detectable apoptosis nor inhibition of angiogenesis. These results show that irreversible cell cycle arrest is sufficient to elicit tumor regression after p53 gene transfer in p53-deficient tumor cells.

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells.

To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins.

Expression of the genes encoding the vasopressin-activated calcium-mobilizing receptor and the dual angiotensin II/vasopressin receptor in the rat central nervous system.

The distributions of two newly discovered receptors, the vasopressin-activated calcium-mobilizing receptor (VACM-1) and the dual angiotensin II/vasopressin receptor (AII/AVP), in the central nervous system (CNS) of the rat were determined using reverse transcriptase-polymerase chain reaction and in situ hybridization. The sequence of the rat VACM-1 cDNA was determined and found very homologous to the rabbit and human sequences. Both VACM-1 and AII/AVP receptor genes were widely expressed in the brain, but differed according to the cell type studied. Glial cells were very faintly labelled. The epithelial cells of the choroid plexuses, the ependymal cells and the pia mater were all labelled. Both genes were most active in neurones throughout the CNS. VACM-1 and AII/AVP receptors were detected in neurones previously shown to possess V1a and V1b vasopressin receptors, and/or the AT1 and AT2 angiotensin II receptors in many brain areas. This was the case for the magnocellular neurones of the supraoptic and paraventricular nuclei of the hypothalamus. We suggest that the VACM-1 and AII/AVP receptors may account for the V2-like responses to vasopressin by these neurones which lack a genuine V2 vasopressin receptor.

The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons.

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.