Dagmara Klopotowska

Transactivation activity of Nur77 discriminates between Ca2+ and cAMP signals

The orphan nuclear receptors Nur77 and Nurr1 are the members of the Nur77 family of transcription factors. We demonstrate that transcription of the Nur77 family genes was upregulated in PC12 cells following incubation with Ca2+ ionophore as well as cyclic AMP (cAMP) analog. On the other hand, cAMP analog induced strong increase, while Ca2+ ionophore induced weak increase in the transactivation activity of Nur77. We found that Nur77 and Nurr1 proteins were expressed in the nucleus following stimulation with cAMP analog but not after stimulation with Ca2+ ionophore. However, expression of Nur77 protein was increased in the cytoplasm of cells treated with Ca2+ ionophore. In conclusion, our results suggest that cAMP-induced and Ca2+-induced processes may differentially regulate activity of Nur77 at the level of translocation of Nur77 protein from the cytoplasm into the nucleus.

Inducibility of doxycycline-regulated gene in neural and neuroendocrine cells strongly depends on the appropriate choice of a tetracycline-responsive promoter

Elucidation of the mechanisms underlying specific receptor activation of neural and neuroendocrine cells will require the establishment of cellular systems that permit the regulation of the expression of the protein of interest. In a tetracycline (Tet)-regulated system, the gene encoding the protein of interest is under the control of a Tet promoter and its transcription is activated in the presence of doxycycline (Dox) by the Tet transactivator rtTA. Acceptable inducibility of the genes expression requires a high level of its expression in the presence of Dox and a minimal basal expression in the absence of Dox. Two Tet promoters are compared here, the original PhCMV*-1 and the second-generation Ptight, with respect to the inducibility of the gene of interest in neuroendocrine and neural cells genetically engineered to express rtTA, namely PC12-Tet-On cells and MB-G-18 cells (mouse brain-derived cells with the phenotype of neuron-restricted precursors). This study demonstrates that the use of Ptight provided a much higher Dox-induced maximal expression in both cell lines, while the basal activities of the two Tet promoters were at similar levels. The additional use of the Tet-controlled silencer (tTS) caused almost complete abrogation of the leakiness of the Ptight promoter and an increase in the inducibility of the regulated gene, but the maximal levels of gene expression driven in the presence of Dox were also markedly reduced.

One-Pot Synthesis of New (1,3-Thiazolo[5,4-b]pyridin-2-yl)benzenediols and Their Antiproliferative Activities against Human Cancer Cell Lines

A one‐pot synthesis of new 4‐(1,3‐thiazolo[5,4‐b]pyridin‐2‐yl)benzene‐1,3‐diols has been described. The compounds were prepared by the reaction of sulfinylbis[(2,4‐dihydroxyphenyl)methanethione] derivatives, with various substituents in the aryl rings, with 2‐chloropyridin‐3‐amines. Their structures were deduced from IR and, 1H‐ and 13C‐NMR spectroscopic, mass spectrometric, and elemental analyses. The antiproliferative properties of some of the products against human cancer cell lines were comparable to those of cisplatin. Structureactivity analysis showed that the presence of hydrophobic substituents in both heterocyclic fused and phenyl rings of the compounds improves their biological effects. Further, an additional OH group in the resorcinol moiety reduced the antiproliferative activity.

Unfavorable effect of calcitriol and its low-calcemic analogs on metastasis of 4T1 mouse mammary gland cancer

Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI-2191 and PRI-2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, real-time PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17β-estradiol and transforming growth factor β (TGFβ) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFβ levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFβ level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFβ and 17β-estradiol levels contributed to the facilitation of metastatic process.

Synthesis of 4-(4-methylidene-4H-3,1-benzothiazin-2-yl)benzene1,3-diols and their antiproliferative activity against human cancer cell lines

One-step synthesis of novel biologically active 4- or 6-(4-methylidene-4H-3,1-benzothiazin-2yl)benzene-1,3-diols is described. The target compounds were prepared by the reaction of aryl-modified sulfinylbis[(2,4-dihydroxyphenyl)methanethione]s with 1-(2-aminophenyl)ethanones. Newly synthesized compounds were characterized by IR, 1H NMR, and mass spectral data. Their antiproliferative potency against human cancer cell lines was determined. Some descriptors were calculated to find the relationship between the structure and activity. It is revealed that the presence of hydrophobic substituents in the benzenediol ring intensifies the biological potency.

Establishment of a cellular model to study TrkC-dependent neuritogenesis

The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (Ptight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.