LDH release from K-562 cells after TNF-α treatment

Jurisic Vladimir 1, Nada Kraguljac2 Bozidar Banicevic2, Gordana Konjevic3, Ivan Spuzic3

1School of Medicine University of Kragujevac, 2Institiuute of Haematology Belgrade, 3Institute of Oncology and Radiology , Belgrade, Serbia, Yugoslavia.

AIM: TNF-a is effects molecule of NK cells and shows diverse effects against malignantly transformed cells, induced cell death in sensitive cells or stimulate growth of some lymphoid cells. The erytroleukemic K-562 cell were originally described by Lozzio and Lozzio from patients in blast transformation and was characterized as "NK sensitive" and usually is a the target for evaluation of cell cytotoxicity. METHODS: Evaluations of TNF-a effects as a single mediator in short-term cultures against K-562 cells was analyzed by enzyme LDH release trough cell membrane simultaneously with evaluation of immunophenotype on gated cell population by flow cytometry. RESULTS: NK cells from healthy persons kill tumor K-562 cells dependent on E :T ratio, but more efficiently than TNF-a alone. The mean values of percentage of LDH release trough cell membrane, calculated from maximal intracellular LDH activity to spontaneous LDH activity from K-562 cells cultured for 2 h with 100 U of TNF-a/ ml did not give significant difference in comparison with mean values of percentage LDH release from K-562 cells cultured without TNF-a (student's t-test, p >0.05). There was no significant increase of LDH release trough cell membrane of K-562 cell preincubated with different concentration of TNF-a (20, 100, 200 U/ml) for 30 min. However, there was significant difference in LDH release, after TNF-a treatment determined after 6 h from K-562 cells in comparison without TNF-a. Flow cytometric analyses shows decrease of CD45 and CD30 antigens expressed on cell membrane after treatment, but significantly increase in expression of CD45RA and GpA antigenes. CONCLUSIONS: Degradation of cell membrane and release of intracellular enzymes as LDH, analyzed in our study is a characteristic for cell membrane damage but nuclear desintegration in late phase of apoptotic mechanism was also mediated by proteolysis. Comparison of NK cell effects with TNF-a suggested that other molecules released from NK cells could participate during early stages of cytotoxicity against K-562 cells and that TNF-a alone was not a major cytotoxic agent. Although, increase of percentage of the LDH release from K-562 cells, preincubated for 30 min. with TNF-a and determined after 6h in comparison with controls, suggested lack of cell membrane, probably by activation of some other molecules in targets which could participate in cytotoxic reactions. As signal transduction pathways involved in apoptotic cell death were poorly understood, the participation of the serine protease enzymes and generation of the second messengers after treatment with TNF-a is possible with modulation expression of some activation antigens on cell membrane indicated complexity of our process and open new research procedure.

For more information, contact

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Molecular Pathology.