alpha-chymotrypsin and Leukemia--Erythroblastic--Acute

The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induce up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B+). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B+ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B+ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B+ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B+ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells.

Topics: Animals; Benzidines; Blood; Cell Differentiation; Cell Line; Chymotrypsin; Culture Media; Dimethyl Sulfoxide; Friend murine leukemia virus; Hemoglobins; Leukemia, Erythroblastic, Acute; Mice; Peptide Hydrolases

We used reagents specific for serine-dependent proteinases to verify that a proteinase of this class is necessary for natural cytotoxicity (NK). NK was inhibited by phenylmethylsulfonylfluoride (PMSF), by diisopropylfluorophosphate (DFP), and by the plasma antiproteinase alpha-1-antichymotrypsin (alpha-1-X), all of which are specific for serine-dependent proteinases. Substrate specificity was then determined on the basis of the specificity of the plasma and fungal anti-proteinases and synthetic alternate substrates that affected NK. alpha-1-X, which inhibits only serine proteinases with aromatic amino acid specificity, blocked NK. Chymostatin, but not other fungal inhibitors, also blocked NK activity. Furthermore, the only synthetic substrates that effectively reduced NK were those derived from aromatic amino acids. The ester derivatives of these substrates inhibited NK better than the amides. NK inhibition with these alternate substrates was also stereospecific, with the L forms twofold more active than the D forms. These reagents did not block initial lymphocyte-target cell binding. Therefore we propose that the "NK-proteinase" is involved in either the initiation of cytolysis, perhaps as part of stimulus and secretion of cytolytic molecules, or in the cascade of events that may lead to the formation of final lytic substance.

Topics: alpha 1-Antichymotrypsin; Amino Acids; Binding, Competitive; Chymotrypsin; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Endopeptidases; Humans; Isoflurophate; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Oligopeptides; Phenylmethylsulfonyl Fluoride; Protease Inhibitors; Serine Endopeptidases; Substrate Specificity

Proteases stimulate mouse erythroleukemia (MEL) cell differentiation and multiplication. The stimulation of differentiation is synergistically increased by low concentrations of dimethyl sulfoxide. Synergism of other low molecular weight inducers with representative proteases, alpha-chymotrypsin and protease V8, was tested. Hemin. hypoxanthine, actinomycin D, aminonucleoside of puromycin, hexamethylene bisacetamide, and 5-azacytidine were also found to act synergistically with this protease to augment MEL cell hemoglobin production, but not cell multiplication. Fatty acids (acetate, propionate, butyrate, isobutyrate, and valerate), prostaglandins A1 and E1, amino acids, and amino acid analogs and metabolites did not act synergistically with chymotrypsin. Some physiologic amino acids were found to be weak inducers. Several of the low molecular weight inducers also acted synergistically with protease V8 in inducing differentiation, and, as with chymotrypsin, did not act synergistically in stimulating cell multiplication. Like chymotrypsin, protease V8 did not act synergistically with butyrate. The earlier finding that proteases, but not low molecular weight inducers, stimulate cell multiplication during the induction of differentiation was confirmed. Carboxypeptidase A also was found to be an inducer.

Topics: Amino Acids; Animals; Butyrates; Cell Differentiation; Cell Division; Cell Line; Chymotrypsin; Dimethyl Sulfoxide; Drug Synergism; Endopeptidases; Erythrocytes; Fatty Acids; Leukemia, Erythroblastic, Acute; Mice; Peptide Hydrolases; Prostaglandins; Serine Endopeptidases

Human erythrocytes with a deficiency in glycophorin A (En(a-) cells) and glycophorin B (S-s-U- and S-s-U+ cells) show significant resistance in vitro to invasion by Plasmodium falciparum merozoites. Treatment of normal erythrocytes with trypsin and chymotrypsin also reduced invasion. Trypsinization of S-s- and En(a-) red cells, a process which removes the T1 peptide of glycophorins A and C, produced cells almost refractory to invasion. The human K562 erythroleukaemia cell line, which also expresses glycophorin A, was not invaded and possible explanations for this result are discussed. It is suggested that determinants carried on the red cell sialoglycoproteins (glycophorins A, B and C), in particular the T1 peptide of glycophorins A and C, play an essential role in attachment and invasion by P. falciparum merozoites. The oligosaccharide components found on this peptide may play a role in the initial binding between red cell and merozoites.

Topics: Animals; Cell Line; Cells, Cultured; Chymotrypsin; Erythrocyte Membrane; Erythrocytes; Genetic Variation; Glycophorins; Humans; Leukemia, Erythroblastic, Acute; Membrane Proteins; Plasmodium falciparum; Sialoglycoproteins; Trypsin