Anti-tumour cytotoxicity of poly(A)-containing messenger RNA isolated from tumour-specific immunogenic RNA

Abstract

Summary.-The transfer of tumour-specific cytotoxicity against a murine fibrosarcoma has been demonstrated in vitro using xenogeneic RNA extracted from tumourcell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA.

Release of soluble “blocking” and “suppressor” factors from normal lymphocytes treated with RNA from spleens of tumour-bearing mice

“…Since the spleen was the source of "blocking" and "suppressor" activities in these studies, and the spleen serves as a source of RNA in our studies, we assumed that the information necessary to transfer "suppressive" responses was contained in our RNA preparations. The transfer of such distinctive suppressor activities would provide additional evidence for the hypothesis that "immune" RNA acts as an informational molecule as others have suggested (Bilello et al, 1976;Dodd et al, 1973;Greenup et al, 1978).…”

“…We have shown that the extracted RNA exhibits a characteristic 3-peak profile (5S, 18S and 28S) on sucrose-density gradients (Dodd et al, 1973;Greenup et al, 1978). The low-molecularweight (4-6S) RNA contains transfer RNA, the second peak (12-20S) comprises mRNA and smaller ribosomal RNA, and the largermolecular-weight (20-35S) RNA accounts for larger ribosomal RNA.…”

“…Credence for these studies is dependent upon the demonstration of antigenic specificity for the transferred response. In a recent report from our laboratory, Greenup et al (1978) using multiple criss-cross experiments with RNA directed at a variety of normal and tumour cell lines, demonstrated that the RNA-transferred cytotoxic response was specific for the eliciting antigen. Similar specificity studies have been made by investigators for many other "positive" types of responses transferred with RNA.…”

“…The methods for immunizing C3H mice against the 4198V tumour cell and the LM cell were as described in a previous paper (Greenup et al, 1978). RNA and lymphocytes were harvested from the spleens of immune animals as described above.…”

“…The procedure for RNA treatment has been described in detail in a previous paper (Greenup et al, 1978). After treatment, the cells were washed and resuspended to a concentration of 5-8 x 106 lymphocytes/ml in RPMI-1640 supplemented with 20% heat-inactivated foetal calf serum, 2 mm L-glutamine, 25 mm HEPES (Sigma Chemical Company, St Louis, Missouri) and 100 jg/ml gentamicin (Schering Corporation, Kenilworth, N.J.).…”