Identification of a thyroid hormone response element in the promoter region of the rat lipocalin-type prostaglandin D synthase (beta-trace) gene.

We have previously reported that mRNA levels for the rat lipocalin-type prostaglandin (PG) D synthase/beta-trace (PGDS) gene, the enzyme responsible for the production of PGD2 in the central nervous system, are regulated by thyroid hormone in vivo. In this study, we describe the identification of a thyroid hormone (T3) response element (T3RE) in the 5'-flanking region of the rat PGDS gene. By radioimmunoprecipitation of genomic fragments using thyroid hormone receptor (TR) protein and specific anti-TR antibodies, gel-shift, foot-printing, mutational analysis, and transactivation assays we have identified a spaced four imperfect direct repeat (DR4) element, GGTTCACTTCAGGGTA (positions -586/-571), which functions as a T3RE when fused to a heterologous promoter. Our results suggest that thyroid hormone regulates the expression of the rat lipocalin-type PGDS gene through this element. Remarkably, the element identified also confers regulation by retinoic acid. Giving the important roles proposed for the PGDS enzyme and its product, PGD2, the major PG in the mammalian brain, the altered expression of the PGDS gene may contribute to the deleterious effects of hypothyroidism in the central nervous system.

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity.

BACKGROUND AND PURPOSE: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity. EXPERIMENTAL APPROACH: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models. KEY RESULTS: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals. CONCLUSIONS AND IMPLICATIONS: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.

Hypothyroidism alters the expression of prostaglandin D2 synthase/beta trace in specific areas of the developing rat brain.

Lipocalin-type prostaglandin D2 synthase is the enzyme responsible for the synthesis of prostaglandin D2, a major prostaglandin in the central nervous system. We analysed the effects of thyroid hormone deprivation on prostaglandin D2 synthase gene expression in the developing rat brain. By in situ hybridization, the strongest prostaglandin D2 synthase mRNA signal was detected in the leptomeninges and choroid plexus. The signal was greatly reduced in the cerebellar interlaminar meninges of hypothyroid rats aged 15 and 25 days. Immunohistochemical studies defined changes in the location of the prostaglandin D2 synthase protein. In control but not in hypothyroid animals, Cajal-Retzius neurons of cortical layer I, and pyramidal cortical plate neurons were intensely stained on postnatal day 5. Conversely, prostaglandin D2 synthase protein levels were higher in neurons of the CA1 and CA3 regions and the dentate gyrus of the hippocampus of hypothyroid animals on postnatal days 5, 15 and 25, and also in subplate neurons on postnatal days 15 and 25. In agreement with the in situ hybridization and northern blotting data, the major difference was found in the cerebellar interlaminar meninges of hypothyroid animals, where the protein was clearly down-regulated on postnatal days 15 and 25. These results show that hypothyroidism causes both age- and region-specific alterations in the expression and location of the prostaglandin D2 synthase during postnatal brain development, probably reflecting a cell-specific regulatory effect of thyroid hormone on the prostaglandin D2 synthase.

Establishment and characterisation of a human carcinoma cell line with acquired resistance to Aplidin.

Aplidin (APL) is a new antitumoral drug from marine origin currently in phase II clinical trials against a wide multiplicity of cancers. As resistance may be, as with other drugs, an important obstacle to the APL therapeutic efficacy, we have established an acquired resistance cellular model by continuous exposure of HeLa cells to the drug. The stably resistant subline generated (HeLa-APL), possessing more than 1000-fold relative resistance to APL than parental cells, did not show crossresistance to a subset of clinically relevant antitumoral agents. In addition, resistance was not related to overexpression of P-glycoprotein or differences in overall drug accumulation. Comparing to parental cells, HeLa-APL cells did not present either significant differences in the growth rate or apparent alterations in the cell cycle distribution. Aplidin induced rapid and persistent phosphorylation of both JNK and p38 MAPKs, resulting in activation of the mitochondrial apoptotic pathway in parental cells, but, notably, in HeLa-APL-resistant cells MAPKs activation only occurred in a slight and transiently manner, failing to activate the above-mentioned apoptotic machinery. These results suggest that sustained activation of JNK and p38 is essential for triggering the apoptotic programme induced by APL and that HeLa-APL cells bypass this apoptotic response by preventing the specific mechanisms that prime and sustain the long-term activation of these signalling cascades. Although far from human tumour physiology in vivo, HeLa-APL cells represent a potentially useful tool in gaining insights into the mode of action of APL, in selecting non-crossresistant APL structural analogues, as well as in investigating and developing methods to prevent resistance to this drug.

Brain-specific prostaglandin D2 synthetase mRNA is dependent on thyroid hormone during rat brain development.

We have previously described several cDNA clones whose expression is affected by thyroid hormone during rat brain development. We now report the identification of one of these, the E2 clone, as the brain-specific prostaglandin D2 (PGD2) synthetase gene. Sequence comparison shows a nearly complete identity between the 356 nucleotides of the E2 clone and nucleotides 403 to 759 of PGD2 synthetase cDNA. The pattern of E2 expression corresponds to that expected for brain specific PGD2 synthetase gene, i.e. the corresponding mRNA is not detected in any other tissue analyzed apart of the brain, and it was present at different levels in all brain regions. Hypothyroidism decreased E2 mRNA concentrations in cerebral cortex and cerebellum. Control of the level of expression of PGD2 synthetase gene may contribute the complex effects of thyroid hormone on brain development and function.

Dexamethasone induces lipocalin-type prostaglandin D synthase gene expression in mouse neuronal cells.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD(2), the main PG in the CNS. PGD(2) is an endogenous sleep inducer, and it is involved in the control of odor and pain responses and body temperature. In addition, PGD synthase transports lipophilic molecules in the subarachnoid space and CSF. By northern and western assays we show that the synthetic glucocorticoid dexamethasone, an inhibitor of PG production in most tissues, induces L-PGDS mRNA and protein in a dose- and time-dependent fashion in mouse neuronal GT1-7 cells. Accordingly, dexamethasone increases cellular L-PGDS enzymatic activity. Dexamethasone induced L-PGDS gene transcription in run-on assays and activated the mouse L-PGDS gene promoter in transiently transfected cells. It is interesting that the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which induces the synthesis of PGs in many tissues, inhibited the increase in L-PGDS expression induced by dexamethasone. In contrast, neither dexamethasone nor TPA affected the expression of cyclooxygenases-1 and -2. Our data demonstrate that dexamethasone induces L-PGDS gene transcription in neuronal cells.

Retinoic acid and 1,25-dihydroxyvitamin D3 inhibit tenascin-C expression in rat glioma C6 cells.

Tenascin-C (Tn-C) is an extracellular matrix protein with growth-, invasive-, and angiogenesis-promoting activities. Tn-C is upregulated during wound healing, tumorigenesis, and other pathological conditions. Highly malignant gliomas with poor prognosis exhibit high levels of Tn-C expression. Here we demonstrate that Tn-C RNA expression in glioma C6 cells is inhibited in a dose-dependent manner by retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (1,25-D3). No additive or synergistic effects were found. Inhibition is maximum 24 hr after RA or 1,25-D3 treatment, prior to a delayed cytotoxic effect starting at day 4-5 of treatment, and correlates with a reduction in the synthesis of Tn-C protein. Tn-C expression is also inhibited, but to a lesser extent by prostaglandin D2 (PGD2). Furthermore, both RA and 1,25-D3, but not PGD2 abolish the induction of Tn-C by the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate. The inhibition of Tn-C expression might be relevant for the anti-cancer activity of RA and 1,25-D3.

Identification and localization of a nucleolin homologue in onion nucleoli.

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.