Cell death of L-929 cells induced by cytotoxic complex Tag7-Hsp70 is analogous to the death of the same cells induced by TNF-α.

The identification and studying the molecular bases of functioning of new cytotoxic agents finds an important implication in developing drugs for fighting with tumors. While investigating the cytotoxic action of protein complex Tag7-Hsp70 which was opened in our laboratory previously we found that Tag7-Hsp70 demonstrated the same specificity in regard to different tumor target cells as it was for classical cytokine TNF-α. L-929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cells within a population and HeLa cells--bad targets representing less than 5% of dead cells after 20 h of incubation with either of the cytotoxic agents. While investigating the action of either TNF-α or Tag7-Hsp70 on L-929 cells we detected two peaks of death: after 3 h and after 20 h. For both cytotoxic agents we observed the first, smaller (13-15%), peak to be eliminated after the addition of caspase inhibitor YVAD-CHO and the second, greater (25-30%), peak to become even bigger in presence of caspase inhibitor. Probably, protein complex Tag7-Hsp70 interacts like TNF-α with a receptor on the surface of tumor cells that results in triggering two alternative mechanisms of programmed cell death: apoptosis and necroptosis.

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.

Nonlymphoid cultured cells possess a system controlling cellular compatibility.

We show that various nonlymphoid cultured cells can activate the production of cytotoxic factors in response to direct contact with cells of a different kind. Accumulation of cytotoxic factors in the medium was detected 1 h after contact of K562 and L929 cells or after contact of L929 cells with purified membranes of K562 cells. TNF-alpha or immunologically related proteins, or both, but not Fas-ligand or lymphotoxin, were also accumulated in membranes of K562 and L929 cells shortly after these cells had been allowed to contact each other. The cytotoxic factors expressed by nonlymphoid cells trigger apoptosis of target cells. These observations strongly suggest that nonlymphoid cells possess molecular mechanisms controlling cellular compatibility.

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.

Inhibitory effect of calf fetuin on the cytotoxic activity of LAK cell-derived factors and tumor necrosis factor.

A protein-inhibiting LAK cell-mediated cytotoxicity was isolated from a LAK cell-conditioned medium. The N-terminal amino acid sequence of this protein appeared to be identical to fetal calf fetuin. Pure isolated fetuin, as well as commercially available preparations of this protein, were shown to inhibit cytotoxic activity of both cytotoxic proteins released by LAK cells and TNF.

Contribution of membrane-associated cytotoxic proteins to cytolysis of L929 and K562 target cells mediated by LAK cells with different phenotypes.

We have demonstrated that the relative contribution of secreted and membrane-associated proteins to the cytotoxicity of LAK cells depended on LAK cell phenotype: the cytotoxicity of CD16+ CD8+ CD3- LAK cells was associated mainly with membrane-bound proteins, and the activity of CD3+ CD8+ CD16- LAK cells was due mainly to secreted soluble proteins. The cytotoxic activity of membrane fractions of LAK cells against cell line L929 was determined by 40-, 32- and 21-kDa proteins for LAK cells bearing NK-specific markers and due to proteins of 34 and 21 kDa in the case of 'CTL-like' LAK-cells.

The role of apoptotic and necrotic processes in cytolysis mediated by LAK cells with different phenotypes.

The role of necrotic and apoptotic pathways in cytolysis mediated by LAK cells was studied. The contribution of necrotic and apoptotic processes to cytolysis depends both on the LAK cells' phenotype and the type of target cells. CD16+/CD8+/CD3- LAK cells induced necrosis of K562 and L929 target cells. The cell death induced by CD3+/CD8+/CD16- LAK cells was found to include features of apoptotic and necrotic processes.

Time-dependent changes of LAK cell phenotypes correlate with the secretion of different cytotoxic proteins.

Human lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) of normal volunteers by interleukin-2 (IL-2) stimulation for 1-8 days. During the first 3 days the surface marker CD16 characteristic for natural killer (NK) cells was expressed and later the CD3 marker characteristic for cytotoxic T cells became predominant. The conditioned media of LAK cells collected after interaction of LAK cells with K562 target cells was chromatographically separated into two cytotoxic fractions: F1 and F2. It was demonstrated that fraction F1 contained cytotoxic proteins having molecular weights of 30 and 40 kDa, and fraction F2 contained cytotoxic proteins having molecular weights of 22, 38 and 75 kDa. The presence of the proteins in each of these two fractions correlated with the phenotype changes of LAK cells: the F2 cytotoxic proteins were characteristic for NK-like cells, and the F1 proteins for cytotoxic T-lymphocyte (CTL)-like phenotypes.