The aim of our work was to evaluate peripheral blood lymphocyte subsets as
in vitro indicators of the received dose of ionizing radiation (biodosimetric markers) in the range of 3-20 Gy and to determine the appropriate time interval, during which a dose-dependent induction of apoptosis occurs upon γ irradiation. In lymphocyte subsets characterized by double color surface immunophenotyping, four-color flow cytometry was used for visualizing cell death-associated increase in superficial phosphatidylserine exposure and cytoplasmic membrane permeability by fluorinated Annexin V
and propidium iodide, respectively. No differences between sham-treated and lethal dose (7 Gy)-irradiated samples were observed upon 6 h cultivation in vitro. Ten and 18 h later, about 50 % of lymphocytes were apoptotic, but only the minority of them was in the late apoptotic phase. The only difference in radioresistance of the CD4+CD8- and CD4-CD8
+ lymphocyte subsets was seen upon 2-day cultivation when huge depletion of intact cells and prevalence of the late apoptotic population became obvious. A dose-dependence study in 16 and 48 h cultures confirmed the effectiveness of major T cell subsets as biodosimetric indicators. On the other hand, the minor CD8+ subset of natural killer (NK) cells has been identified as a radiosensitive lymphocyte population the disappearance of which correlated with the received dose. We demonstrated that the CD3
-CD8+ NK subset can be used as a lethal/sublethal dose discriminator to 16 h cultivation. In addition, our data indicate that two-day cultivation followed by CD3/CD8 expression analysis in an intact lymphocyte population may provide a clue for low dosage biodosimetry.
Advanced interdisciplinary scientific field of tissue engineering has been developed to meet increasing demand for safe, functional and easy available substitutes of irreversibly damaged tissues and organs. First biomaterials were constructed as “two-dimensional” (allowing cell adhesion only on their surface), and durable (non-biodegradable). In contrast, biomaterials of new generation are characterized by so-called three dimensional porous or scaffold-like architecture promoting attachment, growth and differentiation of cells inside the material, accompanied by its gradual removal and replacement with regenerated fully functional tissue. In order to control these processes, these materials are endowed with a defined spectrum of bioactive molecules, such as ligands for adhesion receptors on cells, functional parts of natural growth factors, hormones and enzymes or synthetic regulators of cell behavior, incorporated in defined concentrations and spatial distribution against a bioinert background resistant to uncontrolled protein adsorption and cell adhesion.
The aim of this work was to compare the effect of gamma radiation with sub-low dose-rate 1.8 mGy/min (SLDR), low dose-rate 3.9 mGy/min (LDR) and high dose-rate 0.6 Gy/min (HDR) on human leukemic cell lines with differing p53 status (HL-60, p53 deficient and MOLT-4, p53 wild) and to elucidate the importance of G2/M phase cell cycle arrest during irradiation. Radiosensitivity of HL-60 and MOLT-4 cells was determined by test of clonogenity. Decrease of dose-rate had no effect on radiosensitivity of MOLT-4 cells (D0 for HDR 0.87 Gy, for LDR 0.78 Gy and for SLDR 0.70 Gy). In contrast, a significant increase of radioresistance after LDR irradiation was observed for p53 negative HL-60 cells (D0 for HDR 2.20 Gy and for LDR 3.74 Gy). After an additional decrease of dose-rate (SLDR) D0 value (2.92 Gy) was not significantly different from HDR irradiation. Considering the fact that during HDR the cells are irradiated in all phases of the cell cycle and during LDR mainly in the G2 phase, we have been unable to prove that the G2 phase is the most radiosensitive phase of the cell cycle of HL-60 cells. On the contrary, irradiation of cells in this phase induced damage reparation and increased radioresistance. When the dose-rate was lowered, approximately to 1.8 mGy/min, an opposite effect was detected, i.e. D0 value decreased to 2.9 Gy. We have proved that during SLDR at first (dose up to 2.5 Gy) the cells accumulated in G2 phase, but then they entered mitosis or, if the cell damage was not sufficiently repaired, the cells entered apoptosis. The entry into mitosis has a radiosensibilizing effect.
Migration and proliferation of smooth muscle cells (SMC) were studied in cultures prepared from the aorta of Wistar male rats (170—200 g b.w., 8 weeks old) raised under conventional (CC) or specific pathogen-free (SPF) conditions. In primary cultures, higher movement of cells from explants was found in CC raised donors, namely in samples cultured in serum incomplete medium. In the following subcultures (passage 3—16), the growth curves were steeper and the doubling time shorter in CC type of cultures. The faster growth of SMC population from conventional donors was found to be due to a shorter cell cycle and a higher proportion of dividing cells. As a consequence, the maximum population densities were also higher in the latter type of cultures. The differences in growth, that were dependent on raising conditions, were evident for 16 passages, i.e. 7 months after explantation of cells into culture. The data suggest that breeding conditions may affect the activation of growth of SMC in blood vessels in situ.
Ectopcptidases are widely distributed among various cell systems. Their expression on an appropriate cell type is finely regulated, reflecting the specific functional cell implications and engagement in defined physiological pathways. Protein turnover, ontogeny, inflammation, tissue remodelling, cell migration and tumor invasion arc among the many physiological and pathological events in which cell- surface proteases play a crucial role, both as effector as well as regulatory molecules. It has recently become clear that also non-catalytic effects of membrane-bound proteases are of great importance in some biological regulations. They may generate specific signal transduction intracellularly, after reacting with certain target molecules. They may also play a pivotal role in cell-cell and cell-virus contact and recognition, as well as in binding to the extracellular matrix. This short review provides some insight into the multifunctional mechanisms attributed to cell membrane-bound proteases.