The role of S100A4 protein in anticancer cytotoxicity: its presence is required on the surface of CD4+CD25+PGRPs+S100A4+ lymphocyte and undesirable on the surface of target cells.

S100A4 is a Ca2+-binding protein that performs an important role in metastasis. It is also known for its antitumor functions. S100A4 is expressed by a specialized subset of CD4+CD25+ lymphocytes and is present on those cell's membranes along with peptidoglycan recognition proteins (PGRPs). There, by interacting with major heat shock protein Hsp70, S100A4 plays an important cytotoxic role. The resulting stably formed complex of PGRPs, S100A4 and Hsp70 is required for the identification and binding between a lymphocyte and a target cell. Here, we investigated the S100A4 functions in CD4+CD25+PGRPs+S100A4+ lymphocyte cytotoxicity against target cells. We demonstrated that those lymphocytes do not form a stable complex with the tumor target cells that themselves have S1004A on their surface. That observation can be explained by our finding that S100A4 precludes the formation of a stable complex between PGRPs, S100A4 (on the lymphocytes' surface), and Hsp70 (on the target cells' surface). The decrease in S100A4 level in CD4+CD25+PGRPs+S100A4+ lymphocytes inhibits their cytotoxic activity, while the addition of S100A4 in the medium restores it. Thus, the resistance of target cells to CD4+CD25+PGRPs+ S100A4+ lymphocyte cytotoxicity depends on their S100A4 expression level and can be countered by S100A4 antibodies.

Data of self-made Taq DNA polymerase prepared for screening purposes.

DNA analysis is a key procedure in genetic engineering. Nowadays the analysis is often done by PCR with Taq DNA polymerase. Although the last enzyme price is quite low, demand for numerous analyses results in much money expenditure which are not affordable for many laboratories. In a meanwhile, many screening tasks do not require the highly purified enzyme. Taking into account the enzyme unique properties it makes possible to marginally simplify its production without resorting to costly or lengthy techniques such as column chromatography and/or dialysis. Here the data of routine usage of Taq DNA polymerase prepared according to the protocol developed in our laboratory is presented. The protocol takes only several hours to realize and does not need qualified personnel or expensive equipment. Yet it gives the enzyme preparation suitable for most screening purposes. The isolated Taq DNA polymerase stock can be stored as ammonium sulfate suspension in a refrigerator for prolonged period, not less than 6 months. The working enzyme solution is prepared from the stock suspension on demand, not more than once in a month and can be stored also in a refrigerator.

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1.

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7-Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4(+) and CD8(+) lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.

Interactions and possible functional characteristics of Tag7-S100A4 protein complex.

Peptidoglycane-recognizing protein Tag7 formed a complex with S100A4 (a representative of S100 protein family), the apparent dissociation constants in the absence and presence of Ca2+ were 2 x l0(-8) M and 10(-9) M, respectively. Analysis of fluorescence spectra of hydrophobic fluorescent probe 2-toluidinyl naphthalene-6-sulfonate in the presence of S100A4 and Tag7 proteins showed that extensive area or several sites are involved into the complex formation between these proteins. The formation of Tag7-S100A4 complex had virtually no effect on the role of S100A4 in the regulation of intracellular Ca2+ metabolism. Removal of not only Tag7, but also S100A4 from neutrophil conditioned medium reduced lysis of E. coli cell, while addition of the Tag7-S100A4 complex to the medium restored antibacterial activity.

Comparative analysis of secretion of S100A4 metastatic marker by immune and tumor cells.

S100A4 protein is present in low concentrations (2.1-15.7 ng/10(6) cells) in lymphocyte and neutrophil culture medium. Addition of stimulants to the cells did not lead to an appreciable increase in the content of this protein. The initial content of S100A4 is significantly higher (92-447 ng/10(6) cells) in culture media of highly metastatic KSML-100 adenocarcinoma and M3 and B16 melanoma cells. The release of S100A4 by these cells significantly increased after addition of lymphocytes and Tag7/Hsp70 cytotoxic complex. Repeated injection of antibodies to S100A4 to mice with transplanted M3 melanoma inhibited tumor growth.

Apoptotic and necrotic cytolytic processes induced by membrane associated proteins of LAK cells.

Cytolytic processes induced by membrane-associated proteins of human lymphokine-activated killer (LAK) cells with different phenotypes (CD3+, CD16-, CD8+ and CD16+, CD8+, CD3-) were studied using L929 and K562 types of target cells. Independently of the phenotype of effector cells and the type of target cells, total fractions of membrane proteins induced several different cytolytic processes occurring with different rates and involving different mechanisms of genome fragmentation. The membrane fraction induced, irrespective of the phenotype of LAK cells, mostly apoptotic processes in the L929 line. At the same time, cytolytic processes induced in K562 line differed by the mechanisms of DNA fragmentation. An inhibitor of lysosome activation, NH4Cl, and a Ca(2+)-binding reagent, ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), induced partial inhibition of short-term cytolytic processes (developing within 1-5 h) but did not affect the development of long-term cytolytic processes requiring more than 6-8 h for their development.

Different pathways of the release of cytotoxic proteins in LAK cells.

Human LAK cells were shown to release cytotoxic proteins by both Ca(2+)-dependent and Ca(2+)-independent mechanisms. CD3+ CD8+ CD16- and CD16+ CD8+ CD3- LAK cells were co-incubated with target cells in the presence of 4 mM EGTA. Although EGTA inhibited the exocytosis of cytolytic granules, supernatants obtained were cytotoxic for target cells. Cytotoxicity of CD3+ LAK cells and CD16+ LAK cells was due to cytotoxic proteins with MW 75 (p75), 35 (p35) and 22 (p22) kDa. LAK cells were also shown to release cytotoxic proteins by way of continuous secretion. After co-incubation in the absence of target cells LAK cells can secrete cytotoxic proteins with MW 75 (p75), 55 (p55), 38 (p38), 35 (p35), 25 (p25), 22 (p22) and 17 (p17) kDa.