Marie Audette

The transcription of the intercellular adhesion molecule-1 is regulated by Ets transcription factors

The Ets family of transcription factors comprises several members which are involved to regulate gene transcription. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of effector cells to various target cell types. ICAM-1 is constitutively expressed at different levels in the absence of stimuli in different cell types, and its expression is upregulated by several proinflammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particularly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM significantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the first 176 bp upstream from the translational start site. Electrophoretic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions −158 and −138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM-1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors.

Regulation by retinoic acid of ICAM-1 expression on human tumor cell lines

In a group of four human tumor cell lines comprising one melanoma, one glioma, one teratocarcinoma and one neuroblastoma, the expression of the intercellular adhesion molecule-1 (ICAM-1) was found to be significantly increased following treatment with 10 microM of all-trans retinoic acid. In the melanoma and glioma cell lines HS 294T and HS 683, greater than 90% of the cells reacted with the anti-ICAM-1 monoclonal antibody (mAb) CL203.4 in the absence of treatment. Retinoic acid increased the cell surface expression of the molecule by 2-fold. In the teratocarcinoma and neuroblastoma cell lines, TERA-2 and SK-N-SH, the constitutive expression of ICAM-1 was weak, the percentage of cells stained above the background being less than 25%. Retinoic acid induced ICAM-1 expression in greater than 80% of the cells and increased the levels of expression by 2.5 to 3-fold. Immunoprecipitation studies in biosynthetically labeled cells as well as RNase protection analysis confirmed that retinoic acid treatment increased the amount of ICAM-1 at both the protein and mRNA level. The induction or stimulation occurred within 24 h, was maximal after 4 days and reversible.

RCAS1 is associated with ductal breast cancer progression

RCAS1/EBAG9 (receptor-binding cancer antigen expressed on SiSo cells/ estrogen receptor-binding fragment-associated gene 9), an estrogen-transcribed protein, has been shown to be expressed in a wide variety of cancers, including uterine, ovarian, and lung cancer cells. Soluble and membranous RCAS1 proteins may play a role in the immune escape of tumor cells by promoting T lymphocyte inhibition of growth and apoptosis. In the present report, the presence of RCAS1 was revealed in human ductal breast cancer biopsies by immunohistochemistry. Its cytoplasmic expression was exhibited in cancer cells obtained from tumor biopsies and in breast cancer cell lines. RCAS1 significantly correlated with tumor grade. In addition, RCAS1 was identified in MCF7 culture supernatants. Those observations suggest that RCAS1 is a new marker for breast cancer progression and a possible mechanism for breast cancer immune escape.

Synthesis of spermidine and norspermidine dimers as high affinity polyamine transport inhibitors.

A series of novel spermidine and sym-norspermidine dimers was synthesized by crosslinking the polyamine backbones via alkylation of their secondary amino groups to butyl, trans-2-butenyl, 2-butynyl or p-xylyl bridges. The resulting hexamines behaved as high-affinity antagonists of polyamine uptake, with a relative potency that was dependent on the geometry of the linker structure.

2,2*-Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System*

We have synthesized 2,2′-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with Ki values of 5.0 ± 0.7, 80 ± 31, and 16 ± 3 µM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (Ki = 1.6 ± 0.5 and 2.7 ± 1.1 µM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low Ki toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [3H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 µM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 µM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of specific irreversible inhibitors of the polyamine transporter, which should present advantages over competitive antagonists for an efficient blockade of polyamine transport in tumor cells.

Xylylated dimers of putrescine and polyamines: influence of the polyamine backbone on spermidine transport inhibition.

Dimeric norspermidine and spermidine derivatives are strong competitive inhibitors of polyamine transport. A xylyl tether was used for the dimerization of various triamines and spermine via a secondary amino group, and of putrescine via an ether or an amino group. Dimerization of putrescine moieties potentiates their ability to compete against spermidine transport to a much greater extent than for triamine dimers.

Efficient Transfection of Endothelial Cells by a Double-Pulse Electroporation Method

Primary endothelial cells are largely recognized as hard-to-transfect cells. We have been using a double-pulse electroporation technique to efficiently insert genetic material into human umbilical vein endothelial cell (HUVEC). Previously, this technique has been successfully used on hard-to-transfect monocytic cells. Using a conventional electroporation device, we have tested this protocol on HUVECs and compared it with conventional transfection techniques. The average transfection efficiency was up to 68% as measured by the ability of the cells to efficiently express the red fluorophore of the tdTomato gene. Similar results were obtained in human aortic endothelial cells and human microvascular endothelial cells. This technique does not require any particular expensive device, specific medium, or reagent, and the results we obtained so far exceed those of any other previous protocol. This is therefore an affordable and efficient transfection technique that opens new avenues in vascular endothelial research.

Evidence for a Multistep Model for Eukaryotic Polyamine Transport

One of the most intriguing aspects of polyamine biology is the considerable diversity of their functions in the cell. The involvement of polyamines in such a multiplicity of parallel activities obviously requires mechanisms for the regulation of their concentrations in the various intracellular compartments. Much progress has been made in our understanding of polyamine homeostasis, and it is now clear that antizymes (AZ) are major players in regulating the size of cellular polyamine pools through the feedback inhibition exerted by these proteins on ornithine decarboxylase (ODC) activity and levels, and on polyamine uptake activity (1). However, our current view of how polyamines are distributed throughout the cytoplasm and nucleus after their synthesis is severely limited. The problem of polyamine microcompartmentalization is especially important in eukaryotic cells, where polyamines are expected to simultaneously act in the cytosol (e.g., in ribosomes), in close vicinity of the plasma membrane (e.g., ion channel gating), and in membrane-bound organelles (e.g., nucleus, mitochondria).

Eosinophil Activation Status and Corticosteroid Responsiveness in Severe Asthma

Background: Since eosinophils are implicated in asthma pathogenesis, we investigated whether these cells were activated in severe asthma. Methods: Twenty-six asthmatics with different clinical responses to oral corticosteroid (CS), i.e. sensitive [change in forced expiratory volume in 1 s (ΔFEV1) ≥ 25% after oral methylprednisolone, 40 mg daily, for 14 days, n = 7], resistant (ΔFEV1 ≤ 15%, n = 9) and dependent (≥ 20 mg oral prednisone daily for acceptable asthma control, n = 10), were studied. Results: Calcium ionophore-induced leukotriene (LT) C4 release of purified blood eosinophils was similar in the three groups. Cell incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced ionophore-induced LTC4 release, and this effect was higher in CS-sensitive (5-fold) than in CS-resistant subjects (1.7-fold) (p = 0.02). CS treatment decreased blood eosinophil counts in these two groups of subjects (p ≤ 0.02) and decreased GM-CSF-enhanced LTC4 release in CS-sensitive asthmatics only (p = 0.04). In contrast, despite a high mean daily dose of oral CS (35 ± 8 mg), blood counts of eosinophils from CS-dependent subjects were higher (p = 0.03) and GM-CSF enhancement of LTC4 release was greater (2.8-fold) than in CS-sensitive (2.1-fold) and CS-resistant (1.7-fold) subjects (p = 0.04). Interestingly, serum from CS-resistant subjects reduced GM-CSF enhancement of LTC4 release by eosinophils of CS-sensitive asthmatics (p = 0.001). Conclusions: Eosinophils from CS-dependent asthmatics have an impaired response to CS, whereas serum from CS-resistant subjects contains an inhibitor of eosinophil response to GM-CSF.