AUGMENTATION OF INTERLEUKIN-6 (IL-6) EXPRESSION IN SQUAMOUS CARCINOMA-CELLS AND NORMAL HUMAN KERATINOCYTES TREATED WITH RECOMBINANT ANTINEOPLASTIC PROTEIN (ACP)

A protein purified from Eschericheri coli has previously been shown to have cytotoxic effects on neoplastic cells of several lineages both i n vitro and in vivo. Accordingly, this protein has been named anti-neo plastic protein (ACP). Although ACP kills neoplastic cells by inducing apoptosis, it has negligible effects on various normal cells. In addi tion to the direct cytotoxic effects of ACP on tumour cells, previous studies have shown that in vivo ACP increases tumouricidal activity of cytotoxic lymphocytes. We investigated whether cytokines from host or tumour cells play a part in the enhanced cellular immunity seen in AC P-treated tumour-bearing mice. Growth of normal human keratinocytres ( KC) was not significantly affected by subnanogram amounts of ACP, howe ver ACP dose-dependently killed KHT cells, a murine fibrosarcoma cell line (LD(50)=8X10(4) ng/cell). as well as the human squamous carcinoma cell line COLO-16 (LD(50)=2.5X10(-4) ng/cell). Testing purified A CP on cultures of normal keratinocytes and squamous carcinoma cell lines revealed that ACP could induce both mRNA and protein for interleukin-6 (IL-6). Messenger RN;4 for IL-6 increased dose-dependently 4h after t reatment of COLO-16 squamous carcinoma cells with 10(-4) to 10(-2) ng/ cell ACP. Maximal increment was 50-fold. Interleukin-6 message remaine d elevated up to 24h later in both normal keratinocytes and squamous c arcinoma cultures treated with ACP. Conditioned supernates from these cultures were analysed by ELISA and found to have 4-fold higher levels ,of IL-6 protein than untreated cells after 4h. After 24h, IL-6 did no t increase abo ve the 4h level. Boiling of the ACP preparation showed that the cytokine induction was not due to contaminating lipopolysacch aride. The cytoxic effect of ACP on tumour cells in vitro was not due to IL-6 protein induction since neither recombinant IL-6, nor the othe r proinflammatory cytokines, IL-Ia or Tumour necrosis factor-alpha (TN f-alpha) (0,1-10ng/ml) were able to kill malignant cells. We demonstra ted IL-6 gene induction by ACP in the squamous carcinoma lines as well as in normal KC. This suggests that the in vivo effectiveness of ACP against tumours may be due to stimulatory effects of IL-6 on host immu nity.