Autocrine Survival Factors of a Cytotoxic CTLL-2 Cell Line

Cell survival in multicellular organisms is controlled by numerous cytokines, growth factors, and autocrine survival factors. Autocrine survival factors remain the least studied. The aim of this work was to study the autocrine factors which control survival of a CTLL-2 cytotoxic cell line: isolation and characterization of biologic activity along with physicochemical features of the active molecules have been performed. The conditioned medium of CTLL-2 cells containing autocrine factors was separated by gel filtration into four fractions: A, B, C, and D (according to the order of their efflux from the column). The biological activity of the fractions was tested by the MTT assay with the low density 5 days culture as a cell survival model. The testing of the ability of the fractions to support the cell survival in culture has shown that fractions A and B were active, whereas fractions C and D were not. The presence of a peptide of the molecular mass of 1157 Da in active fractions A and B has been detected by MALDI-TOF-mass-spectrometry. Considerable amount of lactate in fractions A and B, which flowed out from the column along with the peptide, has been detected with an enzymatic lactic acid assay. The lactate concentration in fraction A was 3.72 ± 0.11 mM and it was 0.83 ± 0.06 mM in fraction B. The obtained data suggest that fractions A and B contain supramolecular complexes of the peptide (M 1157 Da) with different lactate content. The peptide in a free form has not been found in the CTLL-2 cell conditioned medium.