Characterization of an inhibitor of cell division released in tumour cell cultures.

We have previously shown that tumour cells may release soluble factors which inhibit the division of a variety of cells. These factors had marked immunosuppressive activity as shown by their ability to inhibit lymphocyte mitogenic responses to lectins, allogeneic lymphocytes and pokeweed mitogen-induced immunoglobulin production. It was postulated that these factors may play an important role in determining the outcome of tumour–host interactions in humans and be the source of immunosuppressive factors in the circulation of patients with cancer. In the present study, characterization of these mitogenic inhibitory factors released into the supernatants of cultured tumour cells was carried out by a number of sequential separation procedures involving affinity chromatography on wheat-germ lectin (WGL), gel filtration on PL-agarose and electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulphate (PAGE–SDS). The factors were identified throughout the separation procedure using inhibition of phytohaemagglutinin (PHA) stimulation of normal lymphocytes as an index of inhibitory function. By these techniques the inhibitory activity was shown to be associated with glycoproteins containing -acetyl-β-D-glucosamine (NAcGlu) and having molecular weights (mol. wt) of approximately 140,000 and over 200,000 daltons. A further small mol. wt peak of 70,000 daltons was observed on PAGE–SDS. pI values of the fractions were 7·4 and 4·2. The activity was destroyed by heating, low pH and repeated freeze-thawing but was resistant to proteolytic enzymes, deoxyribonuclease, ribonuclease and neuraminidase. The active fractions were not associated with proteolytic activity and the inhibition of mitogenic responses was irreversible in nature. These results form the basis of studies to detect this factor in biological fluids and further evaluate its role in the tumour–host relationship.