ISSN 0012�4966, Doklady Biological Sciences, 2013, Vol. 452, pp. 313–315. © Pleiades Publishing, Ltd., 2013.Original Russian Text © A.A. Sheludchenkov, O.D. Kabanova, L.P. Sashchenko, E.A. Romanova, N.V. Gnuchev, D.V. Yashin, 2013, published in Doklady Akademii Nauk, 2013,Vol. 452, No. 2, pp. 230–232.

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1 Exploring new antitumor cytotoxic drugs, under�standing the mechanisms of death of tumor cells uponthe cytotoxic action of these drugs as well as the cytol�ysis of tumor cells upon the action of cytotoxic cells ofimmune system is very important for developing newstrategies for tumor�specific immunotherapy. In thiswork we compared the mechanisms of cytotoxicity ofTag7�Hsp70 complex discovered in our laboratory,with the cytotoxic action of well�known cytokineTNF�α and showed similarity in their cytotoxiceffects.

The first well�characterized natural cytotoxic fac�tors were tumor necrosis factor alpha (TNF�α) andlymphotoxin (LT) [1, 2], which together later wereincorporated in tumor necrosis factor superfamily(TNFSF) comprising about 50 membrane and solubleproteins at the moment [3]. The proteins of TNFsuperfamily perform their functional activity by inter�action with cell surface receptors. These receptors inturn are combined in tumor necrosis factor receptorsuperfamily (TNFRSF) comprising 23 receptors todate [4]. Within this superfamily there is a subclass of

1 The article was translated by the authors.

so�called death receptors. The hallmark of thesereceptors is the existence of a cytoplasmic domainnamed death domain (DD) that takes part in thetransfer of cytotoxic signal [5–7].

For a long time it was thought that interaction ofligands with death receptors can induce only apoptosisbecause it was known that DD can bind with proteinswhich activate specific proteases—caspases—whichthen initiate the apoptotic signaling cascade. However,in 2005 it was discovered that DD can also activateproteins which initiate necroptosis, one of the forms ofprogrammed necrosis. So, one cell surface receptorcan activate two alternative mechanisms of cell death.These mechanisms can’t be induced at the same time:activation of caspases blocks necroptosis becausecaspase�8 cleaves protein kinases RIP1 and RIP3which take part in triggering necroptosis signal trans�duction [8].

Previously in our laboratory it was shown that the pro�tein of innate immunity, Tag7 (PGRP�S/PGLYRP1),can form with heat shock protein 70 (Hsp70) a stableequimolar complex which appeared to be cytotoxic fora number of tumor cell cultures [9]. The same complexis secreted by cytotoxic lymphocytes and suppressesthe growth of tumors transplanted in mice [10]. Butthe precise mechanisms leading cancer cells to thedeath upon the action of this complex are still not clar�ified.

CELLBIOLOGY

Cell Death of L�929 Cells Induced by Cytotoxic Complex Tag7�Hsp70 is Analogous to the Deathof the Same Cells Induced by TNF�α1

A. A. Sheludchenkov, O. D. Kabanova, L. P. Sashchenko, E. A. Romanova, Corresponding Member of the RAS N. V. Gnuchev, and D. V. Yashin

Presented by Academician Georgiev G.P. June 3, 2013

Received June 5, 2013

Abstract—The identification and studying the molecular bases of functioning of new cytotoxic agents findsan important implication in developing drugs for fighting with tumors. While investigating the cytotoxicaction 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 cytok�ine TNF�α. L�929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cellswithin a population and HeLa cells—bad targets representing less than 5% of dead cells after 20 h of incuba�tion with either of the cytotoxic agents. While investigating the action of either TNF�α or Tag7�Hsp70 on L�929cells 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 complexTag7�Hsp70 interacts like TNF�α with a receptor on the surface of tumor cells that results in triggering twoalternative mechanisms of programmed cell death: apoptosis and necroptosis.

DOI: 10.1134/S0012496613050062

Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia

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SHELUDCHENKOV et al.

The aim of this work is to compare the death oftumor cells of cell line L�929 upon the action of pro�tein complex Tag7�Hsp70 with the death of the samecells upon the action of classical cytokine TNF�α forwhich the death receptor structure and the mechanismsexecuting apoptosis and necroptosis are well�known.

In this work we used recombinant human TNF�α

(r�hTNF�α) (Sigma�Aldrich, USA), Tag7�Hsp70 com�plex was made by incubating r�hTag7 and r�hHsp70 for1 h at 37°C as described in [9].

Cells of tumor cell lines L�929 (murine fibrosar�coma), Jurkat (human lymphoblastoma) and HeLa(human cervix carcinoma) were cultured in DMEMsupplemented with 10% fetal calf serum (all fromInvitrogen) at 37°C in a 5% CO2 humidified atmo�sphere; cytotoxic activity was measured by countingdead cells stained by trypan blue as described in [9].

The analysis of the death in populations of differenttumor cell lines upon the action of every of the twocytotoxic agents, the protein complex Tag7�Hsp70and the cytokine TNF�α, after 20 h of their incubationwith cells showed identical specificity of these proteinagents in regard to target cells (TC). On Fig. 1 one cansee that, among different targets, HeLa cells werealmost not sensitive either to the action of Tag7�Hsp70complex or to the action of TNF�α, at the same timeL�929 and Jurkat cells appeared to be good targets foreither of agents under investigation and every agentshowed approximately the same cytotoxicity in regardto TC. But the cytotoxicity of either TNF�α or Tag7�Hsp70 did not exceed 30% which, apparently, wasassociated with the heterogeneity of cell lines and withthe appearance of subpopulations of cells with blockedpathway for cytotoxic signals transfer. The capabilityof tumor cells to avoid immune control is widely dis�

cussed in literature [9, 11]. There are many mecha�nisms for “immunoescape” including the loss of cellsurface receptors as well as the genetic mutationsresulting in loss of functioning proteins which executecell death.

Then we investigated the cytolysis of L�929 cellsupon the action of Tag7�Hsp70 in different timelengths from 1 h to 24 h and compared it with the deathof the same cells upon the action of TNF�α (Fig. 2).The graphic chart representing the dependence ofcytotoxic activity from the incubation time for bothcases doesn’t have linear character and is character�ized by two peaks. The first peak was registered after3 h of incubation, the second—after 20 h. Disconti�nuity of the cytotoxicity and very different rates ofcytolysis may be explained by induction of differentmechanisms of cytotoxicity in different cells inside theheterogenetic population.

It is known that TNF�α causes rapid apoptosis incells with undamaged apoptotic machinery and incells with caspase activity blocked by mutations itinduces necroptosis which is developing for a longertime. We were observing that a population of L�929cell line contained cells dying with different rates uponthe action of TNF�α and it can be supposed that thesecells were dying through apoptotic (3 h) and necrop�totic (20 h) pathways.

For checking this hypothesis we analyzed the deathof L�929 cells upon the action of TNF�α after 3 h andafter 20 h of incubation, in the presence of caspaseinhibitor YVAD�CHO (Fig. 3a). It can be seen that theinhibitor blocked the cytotoxic activity of TNF�α after3 h confirming the developing of apoptosis in thesecells during this time length. It also can be seen thatYVAD�CHO didn’t suppress the cytotoxicity of TNF�

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Fig. 1. The cytotoxic action of TNF�α and Tag7�Hsp70upon tumor cells.Either of the cytotoxic agents, TNF�α and Tag7�Hsp70,was incubated for 20 h with tumor cell cultures of differentlines: L�929, Jurkat, and HeLa. After that the cytotoxicitywas measured, as described in [9].

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Fig. 2. The dynamics of the cytotoxic action of TNF�α andTag7�Hsp70 upon L�929 cells.L�929 cells were incubated with the cytokine TNF�α orwith the protein complex Tag7�Hsp70 for 24 h, the cyto�toxic activity was measured after the same time lengths foreither of the agents.

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CELL DEATH OF L�929 CELLS INDUCED BY CYTOTOXIC COMPLEX 315

α after 20 h but, in contrast, increased the number ofdead cells, indicating that the cells which were previ�ously shown to die through apoptotic pathway, aftersuppressing the caspase activity became dead throughnecroptotic pathway. These results are in accordancewith literature data and with understanding of cyto�toxic action of TNF�α upon cells.

Analogous experiment was done for Tag7�Hsp70complex (Fig. 3b). It is interesting that the data on Fig. 3bare similar to the data on Fig. 3a. In L�929 cells thecytotoxic action of Tag7�Hsp70 in the presence ofcaspase inhibitor was blocked after 3 h and increasedafter 20 h, just as it was for TNF�α. Moreover, anincrease in the number of dead cells after 20 h practi�cally equaled to its decrease after 3 h of incubation withTag7�Hsp70. It gives us grounds to suppose that in thiscase also the inhibition of apoptosis could result innecroptosis. The relationship between the sharp decreasein the number of dead cells after 3 h and its increase after20 h gives reason to suppose that Tag7�Hsp70 complexinteracts with one receptor of cell surface and inducestwo alternative mechanisms of cytotoxicity.

Perhaps, there is a receptor of already known struc�ture in the superfamily of death receptors for whichthe protein complex Tag7�Hsp70 may be a ligand. It isnot excluded that Tag7�Hsp70 complex can be aligand for receptor TNF�R1. Answering these ques�tions is the purpose of our immediate investigations.

ACKNOWLEDGMENTS

This study was performed using equipment of theCore Facilities Center, Institute of Gene Biology, Rus�

sian Academy of Sciences, Moscow, supported by theDepartment of Education and Science of RussianFederation, government contract no. 16.552.11.7067.

This study was supported by the Russian Founda�tion for Basic Recearch, project no. 12�04�01672�a.

This study was supported by the program “Molec�ular and Cell Biology.”

This study was supported by the International Cen�tre of Genetic Engineering and Biotechnology.

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Fig. 3. The cytotoxic action of TNF�α and Tag7�Hsp70 upon L�929 cells preincubated with caspase inhibitor.L�929 cells were preincubated for 1 h with the caspase inhibitor YVAD�CHO added in molarity of 10–3 M, then either the cytok�ine TNF�α (a) or the protein complex Tag7�Hsp70 (b) was added. The cytotoxic activity was measured after 3 h and after 20 h.