Julius Brtko

Amphioxus Postembryonic Development Reveals the Homology of Chordate Metamorphosis

Most studies in evolution are centered on how homologous genes, structures, and/or processes appeared and diverged. Although historical homology is well defined as a concept, in practice its establishment can be problematic, especially for some morphological traits or developmental processes. Metamorphosis in chordates is such an enigmatic character. Defined as a spectacular postembryonic larva-to-adult transition, it shows a wide morphological diversity between the different chordate lineages, suggesting that it might have appeared several times independently. In vertebrates, metamorphosis is triggered by binding of the thyroid hormones (THs) T(4) and T(3) to thyroid-hormone receptors (TRs). Here we show that a TH derivative, triiodothyroacetic acid (TRIAC), induces metamorphosis in the cephalochordate amphioxus. The amphioxus TR (amphiTR) mediates spontaneous and TRIAC-induced metamorphosis because it strongly binds to TRIAC, and a specific TR antagonist, NH3, inhibits both spontaneous and TRIAC-induced metamorphosis. Moreover, as in amphibians, amphiTR expression levels increase around metamorphosis and are enhanced by THs. Therefore, TH-regulated metamorphosis, mediated by TR, is an ancestral feature of all chordates. This conservation of a regulatory network supports the homology of metamorphosis in the chordate lineage.

FUNCTIONAL RETINOID AND THYROID HORMONE RECEPTORS IN HUMAN THYROID-CARCINOMA CELL LINES AND TISSUES

Thyroid carcinomas no longer accessible to radio‐iodide or TSH‐suppressive T4 therapy, due to loss of thyroid‐specific functions, might be sufficiently re‐differentiated by retinoic acid (RA) to be treated by conventional methods again. To help evaluate the feasibility of RA re‐differentiation therapy in thyroid carcinomas, we examined the functionality of RA receptors (RARs/RXRs), central RA signal mediators, in human thyroid‐carcinoma cell lines as model systems. [3H]‐RA binding assays with nuclear extracts from follicular thyroid‐carcinoma cell lines FTC‐133 and ‐238 revealed high‐affinity binding sites for RA. Electrophoretic mobility shift and supershift assays using a DR2 (“direct repeat” 2) RA response element demonstrated DNA‐binding of RARα, RARγ, RXRα and RXRβ in nuclear extracts of FTC‐133 and anaplastic HTh74 cells. Use of a DR5 RA response element revealed no difference in DNA binding. In supershift assays with a DR4 T3 response element, we found DNA‐binding by TRα1, TRα2, and TRβ. Northern‐blot analysis showed low expression of RXRβ mRNA in FTC‐133 and of TRα1 mRNA in FTC‐133 and FTC‐238 cells. Using RT‐PCR, we detected mRNA for RARα, RARβ, RARγ, RXRα, and RXRβ in the 4 cell lines and in human thyroid‐carcinoma samples. RARβ mRNA was reduced in FTC‐238 cells and RXRβ mRNA was decreased in anaplastic C643 cells and 9 of 12 tumor samples. Differential RA regulation of RA‐receptor‐mRNA expression was observed in the various cell lines. Thus, RA and T3 nuclear receptors are present in thyroid‐carcinoma cell lines or tissues, albeit with cell‐line and tumor‐dependent variations; in the cell lines, they were shown to be functional with respect to DNA and/or ligand binding. Int. J. Cancer 76:368–376, 1998.© 1998 Wiley‐Liss, Inc.

Altered Function of Peripheral Organ Systems in Rats Exposed to Chronic Mild Stress Model of Depression

Abstract1. In depression, psychiatric symptoms are frequently associated with impaired cardiovascular function and perhaps also increased risk for cancer diseases. Pathophysiological basis of this comorbidity is not clearly understood. Molecular events involved, particularly factors modified by chronic stress exposure, may only be evaluated in animal models of depression.2. Present experiments were aimed to study parameters related to cardiovascular system (tyrosine hydroxylase (TH) gene expression in adrenal glands) and carcinogenesis (retinoic acid receptors in the liver) in the chronic mild stress model of depression.3. Chronic mild stress induced a rise in adrenal TH gene expression in both male and female rats. Gender dependent changes were found in retinoic acid receptor binding with stress-induced activation in females but not males. Ovariectomized animals exhibited higher retinoic acid receptor binding, slightly elevated TH mRNA levels and failed to respond to chronic mild stress exposure with further increase in TH mRNA levels. Similarly, chronic mild stress induced an anhedonic state manifested by decreased sucrose preference in control but not ovariectomized rats.4. Presented data document that central neurochemical and behavioral changes in animals exposed to chronic mild stress model of depression are associated with changes in adrenal TH gene expression and with gender dependent changes in retinoic acid receptor status in the liver. Such alterations may participate in the development of pathological changes and could participate on increased risk for cardiovascular and oncologic comorbidity in depressive patients.

Expression of nuclear retinoic acid receptor in peripheral blood mononuclear cells (PBMC) of healthy subjects

In vertebrates, both nuclear all-trans and 9-cis retinoic acid receptors (RAR and RXR) belonging to the steroid/thyroid/retinoid nuclear receptor superfamily play a crucial role in the vitamin A action. Qualitative analysis of all known RAR or RXR subtypes in both pooled and non-pooled peripheral blood mononuclear cells (PBMC) from healthy human subjects has been performed by reverse transcription and polymerase chain reaction (RT-PCR). Our data, based on qualitative RT-PCR analysis has shown that human PBMC are capable to express RAR alpha, RAR gamma, RXR alpha, and RXR beta.

Antileukemic activity of 4-pyranone derivatives

In a search for new compounds possessing antitumor activity, we examined the effects of a group of oxygen containing heterocyclic derivatives on L1210 murine leukemia cell growth. Several 5-hydroxy-2-hydroxymethyl-4-pyranone derivatives were tested in a growth assay employing a human leukemia K562 cells line. IC50 was extrapolated from the growth inhibition curves at compound concentrations ranging from 0.1 to 100 microM. The halogen derivatives of 5-hydroxy-2-hydroxymethyl-4-pyranone inhibited L1210 cell growth in suspension culture after 96 hr incubation in the following order: 5-hydroxy-2-iodomethyl-4-pyranone (IC50 3.15 microM) > 6-bromo-2-bromomethyl-5-hydroxy-4-pyranone (IC50 3.40 microM) > 6-bromo-5-hydroxy-2-hydroxy-methyl-4-pyranone (IC50 3.75 microM) > 2-bromomethyl-5-hydroxy-4-pyranone (IC50 4.30 microM) > 5-benzyloxy-2-chloromethyl-4-pyranone (IC50 5 microM) > 6-bromo-2-chloromethyl-4-pyranone (IC50 13.50 microM) > 6-chloro-2-chloromethyl-5-hydroxy-4-pyranone (IC50 18 microM) > 2-chloromethyl-5-hydroxy-4-pyranone (IC50 20 microM). The compound, 5-hydroxy-2-hydroxymethyl-4-pyranone has no effect on L1210 cell growth. These results suggest that 5-hydroxy-2-hydroxymethyl-4-pyranone derivatives might represent a new class of compounds with antileukemic activity.

Effect of selenite and selenate on rat liver nuclear 3,5,3′-triiodothyronine (T3) receptor

The present study was undertaken to investigate the effects of selenite (SeIV) or selenate (SeVI) on nuclear T3 receptors of rat liver. Selenite at 0.1 μM (p<0.01) inhibited the T3 specific binding to rat liver nuclear receptors. The specific binding of the T3 receptor was fully restored when even 1.0 μM selenite was separated from the T3 receptor by gel filtration. No inhibitory effect of selenite (up to 100 μM) on the T3 binding to nuclear receptor was found in the presence of 1.0 mM dithiothreitol. The rate of dissociation of the T3-nuclear receptor complex was effectively increased by 0.1 μM selenite. Selenate up to 1 mM as well as sulfite or sulfate up to 0.1 mM did not exert an inhibitory effect on T3 receptors. The results based on the in vitro experiments suggest that the selenium in the form of selenite may reversibly affect the T3 binding on the receptor molecule.

Relationship between antifungal activity and hydrophobicity of kojic acid derivatives

Twenty three kojic acid derivatives were tested in the agar diffusion test against twenty five dermatophytic fungi. The inhibitory effects at equal doses (10 mg/L) were correlated with the 1-octanol — water partition coefficients of the compounds. An improvement in antifungal activity can be achieved by increasing the hydrophobicity of the compounds.

Expression, protein stability and transcriptional activity of retinoic acid receptors are affected by microtubules interfering agents and all-trans-retinoic acid in primary rat hepatocytes.

Cellular signaling by glucocorticoid receptor and aryl hydrocarbon receptor is restricted by microtubules interfering agents (MIAs). This leads to down-regulation of drug metabolizing enzymes and drug interactions. Here we investigated the effects of all-trans-retinoic acid (ATRA) and MIAs, i.e. colchicine, nocodazole and taxol on the regulation of retinoic acid receptor (RAR) genes in primary cultures of rat hepatocytes. ATRA (1microM) down-regulated RARalpha and RARgamma mRNAs (decrease 23% and 41%, respectively) whereas it up-regulated RARbeta mRNA (4.3-fold induction). All MIAs diminished the expression of RARs in dose-dependent manner; the potency of MIAs increased in order NOC<COL<TAX and the extent of inhibition increased in order RARalpha<RARgamma<RARbeta. The levels of RARalpha protein were decreased by both MIAs and ATRA. The effects of ATRA were reversed by proteasome inhibitor MG-132, implying ligand-dependent RARalpha degradation. In contrast, the effects of MIAs were proteasome-independent and decrease in RARalpha protein content was due to RARalpha gene down-regulation. We monitored transcriptional activity of RARalpha. For this purpose, we measured catalytic activity of trans-glutaminase-target gene of RARalpha. trans-Glutaminase activity was increased by ATRA (1.23-fold increase) and decreased by colchicine (decrease 51%). Co-treatment with proteasome inhibitor MG-132 partly reversed inhibitory effect of colchicine, and it further augmented the increase of trans-glutaminase activity by ATRA. We have also observed decrease of RARalpha protein level and inhibition of RARs mRNAs expression in HeLa cells by MIAs. In conclusion, our data indicate that microtubules play the role in regulation of RARs activity and expression. Our data are the first report on the effects of ATRA and MIAs on RARs regulation in quiescent cells.

A comparison of the effects of tributyltin chloride and triphenyltin chloride on cell proliferation, proapoptotic p53, Bax, and antiapoptotic Bcl-2 protein levels in human breast cancer MCF-7 cell line

In recent years it was disclosed, that numerous organotin(IV) derivatives have remarkable cytotoxicity against several types of cancer cells. The property to inhibit cell growth makes these compounds promising for antitumor therapy, as the clinical effectiveness of cisplatin is limited by drug resistance and significant side effects. Tributyltin and triphenyltin are known as endocrine disruptors. Moreover, the compounds exert their toxicity in mammals predominantly through nuclear receptor signaling. Here we present the effects of tributyltin chloride (TBT-Cl) and triphenyltin chloride (TPT-Cl) on cell proliferation, expression of proapoptotic p53, Bax, and antiapoptotic Bcl-2 proteins in human breast cancer MCF-7 cell line. Dose and time dependent (24, 48 and 72 h) cell expositions have demonstrated TBT-Cl as more effective in inhibiting MCF-7 cell proliferation than TPT-Cl. Short time treatment with TBT-Cl displayed marked stimulation of p53 protein expression when compared to TPT-Cl. Both organotin compounds displayed similar mild enhancement of Bax protein expression. The 24h exposition of TPT-Cl induced substantial diminution of Bcl-2 protein expression in comparison with both, untreated cells and TBT-Cl treated cells. Our observations indicate that TBT-Cl and TPT-Cl have different antiproliferative potency and distinct impact on expression of apoptosis marker proteins.

MNU-induced carcinogenesis of rat mammary gland: Effect of thyroid hormone on expression of retinoic acid receptors in tumours of mammary gland

The rat model of N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas is well-established animal model for breast cancer. This study was carried out to investigate whether hypothyroid (thyroidectomy or PTU treatment) or hyperthyroid status of female rats would affect MNU-induced mammary carcinogenesis with specific focus on both retinoid and rexinoid receptor expression in mammary tumours. Application of PTU before and during MNU-induced mammary gland carcinogenesis yielded in a marked decrease of the number and volume of tumours per animal, however, there was no effect of hypothyroid state in thyroidectomized rats as well as hyperthyroid state concerning the number and volume of tumours. Mammary tumours of in euthyroid group of MNU animals showed that there was no tumour, in which all of subtypes of retinoid and rexinoid receptors were expressed. A different pattern of expression of retinoid or rexinoid receptors was found either in MNU-induced mammary carcinomas in both hypothyroid and hyperthyroid rats.