Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells.

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.

Effects of isradipine, an L-type calcium channel blocker on permanent and transient focal cerebral ischemia in spontaneously hypertensive rats.

Permanent or transient focal cerebral ischemia was induced in spontaneously hypertensive rats (SHR) using the intraluminal filament method. Successful occlusion of the middle cerebral artery (MCA) was achieved using 4/O filaments (terminal diameter 0.20-0.25 mm) coated with poly-L-lysine. The L-type calcium channel blocker isradipine (2.5 mg/kg) administered subcutaneously 30 min following permanent MCA occlusion significantly reduced the volume of ischemic brain damage in the cerebral hemisphere (25%; P = 0.0001), cerebral cortex (18%; P = 0.0034), and caudate nucleus (33%; P = 0.0002) when assessed at 24 h post-MCA occlusion. Isradipine did not affect the functional deficit (measured using a subjective neurological scoring system) induced by MCA occlusion. In SHR undergoing transient (2 h) MCA occlusion isradipine administered 30 min post-MCA occlusion produced a significant reduction (47%; P = 0.001) in hemispheric infarct volume, whereas isradipine administered at the onset of reperfusion did not confer any significant neuroprotection. No change in functional deficit was seen with isradipine with either dosing paradigm at 24 h post-MCA occlusion. These results demonstrate that the intraluminal filament method of MCA occlusion can be used in the SHR strain and also substantiates the neuroprotective efficacy of isradipine in SHR models of permanent and transient focal cerebral ischemia.

Identification of a zinc-finger gene at 6q25: a chromosomal region implicated in development of many solid tumors.

We have used a rat model of epithelial ovarian cancer to identify a gene that shows decreased or lost expression in independently transformed rat ovarian surface epithelial cell lines compared to the normal progenitor cells. Hence, we refer to this gene as Lot-1 (Lost on transformation 1, GenBank accession no. U72620). Here, we report the cloning of the likely human homologue and its initial characterization. The deduced amino acid sequences of the cDNAs for rat and human LOT-1 (GenBank accession no. U72621) contain seven zinc finger motifs of the C2H2 type as well as proline and glutamine rich areas. The genes share 76.4% identity at the nucleotide level, 67.7% at the amino acid level and 85.5% within the seven zinc finger motifs. LOT-1 is ubiquitously expressed in normal human tissues but was not expressed in four of 11 (36%) human ovarian cancer cell lines or spontaneously transformed human ovarian surface epithelial cells. The human gene maps to chromosome 6 at band q25. We show that there is a 38% incidence of allelic loss at this chromosomal location in human ovarian cancers. This chromosomal region has also been implicated in the genesis of breast, kidney, and pleural mesothelial cancers. We suggest that this newly identified gene is not only of intrinsic interest as a ubiquitously expressed probable transcription factor but is a plausible candidate for the tumor suppressor gene which likely resides in the region of chromosome 6 defined by band q25.

Comparison of some effects of paroxetine with amitriptyline on the cardiovascular system in animals.

The effects of intravenous infusions of paroxetine, a novel inhibitor of 5-hydroxytryptamine (5HT) uptake, and of the tricyclic antidepressant, amitriptyline, on the cardiovascular system have been compared in the conscious rabbit and in the anaesthetised cat. As judged by the dose required to produce changes in ECG waveform (including PR and QTc intervals) and disorders of heart rhythm, paroxetine was less cardiotoxic than amitriptyline in both species. Thus, paroxetine has the advantage over amitriptyline of being less toxic to the cardiovascular system which could constitute a considerable advantage in clinical use particularly as other work has shown it to be more potent than amitriptyline in tests for antidepressant activity.

Activity of tricyclic nucleoside 5'-phosphate in model systems of human ovarian cancer.

Tricyclic nucleoside 5'-phosphate (TCN-P) was evaluated in two models of human ovarian cancer. TCN-P reduced both colony number and volume in clonogenic assays employing human ovarian cancer cell lines. TCN-P cytotoxicity depended on the concentration, exposure duration and cell line studied, but not on cell line plating efficiency or growth rate in soft agarose. Comparison of experimental IC50 concentrations for 1 hour or continuous TCN-P exposure with reported clinically relevant concentrations suggests that therapeutic TCN-P levels are more likely to be achieved by continuous infusions. Cell lines and sublines with resistance to several standard chemotherapeutic agents acquired both in vivo and in vitro were at most 2.6-fold cross-resistant to TCN-P with 1 hour drug exposure. Cross-resistance was not evident with continuous TCN-P exposure. Intermittent bolus TCN-P (100 mg/kg/d X 5) was ineffective in an in vivo xenograft model of human ovarian cancer. These data suggest that TCN-P is most likely to be clinically effective against ovarian cancer, and may be non-cross-resistant with several standard agents, if administered by continuous infusion. Preclinical evaluation of new agents, such as TCN-P, in these experimental models may provide information useful in subsequent clinical trials.

Electron microbeam analysis of calcium distribution in the ciliated protozoan, Spirostomum ambiguum.

Electron microprobe analyses of calcium distribution in the ciliated protozoan, Spirostomum ambiguum, indicated several calcium rich sites. One site was an endoplasmic distribution of calcium coincident with phosphorus which corroborates previous findings of hydroxyapatite deposits within Spirostomum. These apatite deposits were distributed throughout the endoplasm, but not within the nuclei or the contractile vacuole. Calcium was also detected within the cortical region. Cortical calcium was in greater concentration in the anterior portion of the organism and decreased towards the posterior end (region containing the contractile vacuole). Phosphorus and potassium were also detected as gradients from the anterior end, whereas magnesium was detected in the same density throughout the cortical region. Line scans of cortical regions suggested (1) distributions of calcium within mitochondria and/or vesicles, and (2) calcium associated with bundles of microfilaments.