Varying effects of EGF, HGF and TGFß on formation of invadopodia and invasiveness of melanoma cell lines of different origin.

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor ß (TGFß) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastases. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in metastatic cell lines treated with TGFß. Our findings indicate that the effects of stimulation with EGF, HGF and TGFß on melanoma cell invasiveness could depend on melanoma cell progression stage.

Proteome of spleen CD4 lymphocytes in mouse preimplantation pregnancy.

Pregnancy exerts profound impact on female immune system. The first signs of pregnancy recognition by immune system are observed even before implantation. The most visible effects are present in the local compartment, i.e. in uterine draining lymph nodes and the decidua, while peripheral changes are less obvious. In our recent paper we indicated that costimulation phenotype of APCs in spleens of female mice during the preimplantation period of pregnancy differs from mice in pseudopregnancy. However, the effect of differential costimulation in the context of the T lymphocyte function at periphery in early pregnancy is still unknown. For that reason, we decided to investigate global protein expression in splenic CD4(+) lymphocytes in order to identify and validate the most important biomarkers characteristic for the preimplantation period of pregnancy at periphery. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were utilized to analyze the protein expression pattern of magnetically sorted CD4(+) lymphocytes from spleens of pregnant and pseudopregnant females at 3.5 days after mating. The first goal of this study was to create a 2-DE map of the splenic CD4(+) T cells of pregnant mice. As a result, 106 protein spots from 373 were identified using MS. The comparison of lymphocyte protein patterns between pregnant and pseudopregnant mice depicted differential expression of 11 identified proteins belonging to the group of proteins involved in cytoskeletal structure, cell motility and metabolism. Profoundly diminished expression of cofilin-1, F-actin capping protein subunit alpha and malate dehydrogenase proteins in lymphocytes of pregnant mice indicates that preimplantation pregnancy could change the activation state of peripheral CD4(+) lymphocytes.

Induction of fat cell necrosis in human fat tissue after treatment with phosphatidylcholine and deoxycholate.

BACKGROUND: Injections with phosphatidylcholine- and deoxycholate-containing substances are used to treat localized fat accumulation and lipomas. It is believed that the injected substances induce fat cell destruction with subsequent acute panniculitis followed by a repair process of the treated fat tissue. OBJECTIVES: We investigated whether necrosis or apoptosis of fat cells was induced by the injected substances. METHODS: Samples of fat tissue of lipoma were collected at various times after injection and evaluated by light and electron microscopy, by immunostaining for active caspase-3 and antideoxyribonuclease I, in situ end-labelling (TUNEL staining), and biochemical caspase-3 assays. RESULTS: Light and electron microscopy showed fat cell necrosis in all areas of the treated lipomas. Low levels of active caspase-3 indicated the absence of apoptosis. CONCLUSIONS: Injection of the lipolytic substances phosphatidylcholine and deoxycholate leads to fat cell necrosis rather than apoptosis. However, additional studies evaluating different dosing and further time points after treatment are necessary.

Subcellular distribution and expression of cofilin and ezrin in human colon adenocarcinoma cell lines with different metastatic potential.

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines - parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells. In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.