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Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of C1s.

The amount of Forssman glycolipid (GL-5) was investigated in two clones of Nil hamster cells grown either in monolayer or in spinner culture. GL-5 assayed by the complement-fixation inhibition test increased with increasing cell density in the monolayer. However, cells grown in spinner cultures failed to show the density-dependent response in both clones examined. Cells transformed by hamster sarcoma virus did not show the density-dependent increase of GL-5, even in cells grown in monolayer. The effect of transfer from confluent to sparse cultures on the amount and the synthesis of GL-5 was also examined. It is suggested that the GL-5 that accumulates in cells during confluency is diluted into the daughter cells and that the decrease of the Forssman lipid does not precede cell division.

Oncogenic transformation of mouse 10T 1/2 fibroblasts induced upon exposure to X-ray or N-methyl-N'-nitro-N-nitrosoguanidine was suppressed if lipopolysaccharide (LPS) was present in the culture medium. The suppressive effect of LPS was exerted within 24 h after irradiation. Suppression was dependent on the concentration of LPS added and LPS (2 micrograms/ml) derived from Salmonella minnesota R595 reduced the number of transformed type III foci per dish from 0.39 to 0.15. Indomethacin (1 to 30 microM) further enhanced the effect of LPS in a dose-dependent manner.

Glycolipid synthesis was investigated in secondary hamster embryo cells and in two Nil (hamster) clones at 24-hr intervals following infection with the hamster sarcoma virus. In all cases lower levels of complex glycolipids were being synthesized by 14 days postinfection. The time courses for these changes, however, were complex and characteristic for each cell type. Ahigher rate of Forssman antigen synthesis was found in infected secondary hamster fibroblasts as compared to uninfected cells at one point during the transition to transformation. In some cases the changes in glycolipid pattern were found to precede changes in morphology and glucose uptake and in other cases they were found to follow these changes.