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3 Archivum Immunologiae et Therapiae Experimentalis

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The cGMP synthesis and PKG1 expression in murine lymphoid organs

100%

Kurowska E. , Kobialka M. , Ziolo E. , Strzadala L. , Gorczyca W. A.

Archivum Immunologiae et Therapiae Experimentalis

2002

tom 50

nr 4

289-295

Numerous reports indicate that cyclic 3',5' guanosine monophosphate (cGMP) is involved in the regulation of immune processes. However, the mechanisms responsible for the synthesis of this nucleotide and its signaling pathways in immune cells are still not well recognized. The aim of our study was to establish: 1) which form of guanylyl cyclase synthesizes cGMP in murine lymphoid organs and 2) whether the same organs express the isoforms PKG1alpha and/or PKG1beta of protein kinase G, known as a possible target for synthesized cGMP. Cells isolated from thymus, lymph nodes, and spleen were treated with activators (SNP, ANP, CNP, STa) of soluble or particulate cyclases. Sodium nitroprusside (SNP) elevated intracellular cGMP 2-fold in thymic and lymph node cells and about 10-fold in spleen cells. Atrial natriuretic peptide (ANP) caused modest but statistically significant increases of cGMP in cells of all the organs. Additionally, spleen cells elevated their cGMP content about 2-fold in response to C-type natriuretic protein (CNP). In cellular homogenates of all the analyzed organs, the antibody anti-PKG1beta stained the 78 kDa band corresponding to the molecular mass of PKG1. Only homogenates of spleen cells were stained by the antibody recognizing PKG1alpha. Our results indicate that in all the investigated organs, cGMP may be synthesized mainly by soluble guanylyl cyclases in response to nitric oxide. The modest increase of cGMP upon stimulation by ANP suggests that in all these organs either exist only a small subpopulation of cells that express particulate cyclase GC-A or GC-A is expressed at very low level. In spleen cells, however, cyclase GC-B appears to be the more active enzyme. Elevated cGMP concentration may in turn activate PKG1beta in thymus, lymph node, and spleen cells and also PKG1alpha in spleen cells.

Changes in glucocorticoid-induced apoptosis and in expression of Bcl-2 protein during long-term culture of thymic lymphoma

100%

Kobzdej M. , Matuszyk J. , Ziolo E. , Strzada?a L. , Strzada?a L.

Archivum Immunologiae et Therapiae Experimentalis

2000

tom 48

nr 1

43-46

Lymphomagenesis is a multistep process progressively freeing transformed thymocytes from external regulatory signals, i. e. thymic developmental program controlling growth, differentiation or apoptosis. Here we report that cells of thymic lymphoma overexpressing Ras/Raf proteins, initially resistant to TCR-dependent apoptosis but sensitive to dexamethasone- and etoposide-induced cell death, became insensitive to dexamethasone after long-time culture. That transition correlated with a strong increase in the expression of the anti-apoptotic Bcl-2 protein. Interestingly, lymphoma cells were still sensitive to p53-mediated apoptosis induced by etoposide. It suggests that the anti-apoptotic activity of Bcl-2 is correlated with a resistance to glucocorticoid-induced apoptosis but not to p53-mediated apoptosis. The sequence of mutations in the process of lymphomagenesis seems to be composed of at least 3 main hits which equip the cells with independence from external mitogenic signals (activation of Ras/Raf), resistance to inducers of apoptosis (activation of Bcl-2) and generation of cellular heterogeneity (deletion of p53) important in tumor progression.

In vitro photodynamic therapy with chlorin e6 leads to apoptosis of human vascular smooth muscle cells

86%

Wawrzynska M. , Kalas W. , Bialy D. , Ziolo E. , Arkowski J. , Mazurek W. , Strzadala L.

Archivum Immunologiae et Therapiae Experimentalis

2010

tom 58

nr 1

67-75

Percutaneous coronary intervention has become the most common and widely implemented method of heart revascularization. However, the development of restenosis remains the major limitation of this method. Photodynamic therapy (PDT) recently emerged as a new and promising method for the prevention of arterial restenosis. Here the efficacy of chlorin e6 in PDT was investigated in vitro using human vascular smooth muscle cells (TG/HA-VSMCs) as one of the cell types crucial in the development of restenosis. PDT-induced cell death was studied on many levels, including annexin V staining, measurement of the generation reactive oxygen species (ROS) and caspase-3 activity, and assessment of changes in mitochondrial membrane potential and fragmentation of DNA. Photosensitization of TG/HA-VSMCs with a 170 M of chlorin e6 and subsequent illumination with the light of a 672-nm diode laser (2 J/cm2) resulted in the generation of ROS, a decrease in cell membrane polarization, caspase-3 activation, as well as DNA fragmentation. Interestingly, the latter two apoptotic events could not be observed in photosensitized and illuminated NIH3T3 fibroblasts, suggesting different outcomes of the model of PDT in various types of cells. The results obtained with human VSMCs show that chlorin e6 may be useful in the PDT of aerial restenosis, but its efficacy still needs to be established in an animal model.