The human colon cancer cell line HT-29 produces a growth factor (CRDGF; Mr = 25,000) which inhibits EGF binding to a wide variety of different normal and tumoral cell types in culture. Scatchard analysis of EGF binding shows that CRDGF induces a decrease in EGF receptor affinity. In contrast, EGF binding to any of the human colorectal cancer cell lines tested, i.e., HT-29, HT-29 (clone D4), HRT-18 or CAL-14, remains unaltered in the presence of exogenous CRDGF. However, the inhibitory effect of CRDGF becomes apparent on HT-29 cells after overnight exposure of these to suramin (at 37 degrees C). A short exposure to suramin (1 hr at 4 degrees C) or a mild acid washing of HT-29 cells can partially restore the inhibitory activity of CRDGF. These observations suggest that the action of suramin results in an unmasking of substantial levels of CRDGF receptors on HT-29 cells. Scatchard analysis of EGF binding on suramin-treated HT-29 cells shows that CRDGF inhibits EGF binding by decreasing EGF receptor affinity, as previously observed with the non-colonic cell types. A similar unmasking of CRDGF receptors is observed when the other colorectal cell lines are exposed to suramin. These results provide evidence for a model in which the colorectal cell lines have the property of secreting a unique growth factor that binds to its receptor by an autocrine mechanism.
Products secreted by HT-29 human colonic adenocarcinoma cells (DMEM-HT-29) mediated strong suppressive activity of in vitro lymphoproliferative responses to several mitogens. In vivo administration of DMEM-HT-29 both inhibited the afferent limb of delayed-type hypersensitivity against the Mc FiFi2(s) syngeneic fibrosarcoma and delayed the rejection of these tumor cells by immunized animals. Transfer experiments prior or after cell fractionation did not demonstrate suppressor cells induced by DMEM-HT-29. This suggests that DMEM-HT-29 produces its effect by directly interacting with macrophage and/or T cells at the sensitization stage of the antitumor immune response.
The generation of superoxide anion (O2-) by human blood monocytes in response to stimulation by either phorbol myristate acetate (PMA) or opsonized Zymosan was greatly enhanced (range: 100-200% according to donor) by prior exposure of the peripheral blood mononuclear cells (PBM) to human colonic adenocarcinoma cells (HT 29 line) or their conditioned culture medium (DMEM-HT 29). This priming effect was observed after 5 hr and persisted for up to 15 hr of contact between PBM and endotoxin-free DMEM-HT 29. Beyond this time, primed monocytes gradually lost this ability. However, they maintained a higher capacity (about 100%) to produce O2- when compared to controls. DMEM-HT 29-induced monocyte priming requires that the tumor-active substance(s) act(s) on 2 target cells: first, on non adherent mononuclear cells (NA-PBM) to induce cytokine production and, second, on the monocyte itself. Priming activity was also found in conditioned medium from FR3T3 embryonic fibroblasts but not in conditioned medium from HT 29 repolarized cells (by culture in glucose-free medium) or from non-tumorous human colonic mucosa explants.
We have studied the effect of pertussis toxin (PT) on in vivo priming of T lymphocytes to insulin. Mice were immunized with bovine insulin in complete Freund's adjuvant and antigen-specific DNA synthesis was measured in lymphoid cell suspensions from lymph nodes and spleens. Insulin-specific response was greatly enhanced both in spleen and lymph nodes if mice were given PT at the time of immunization. Mice given PT presented 3 times more cells in spleen and 4 times less in lymph nodes. However, the major antigen-specific response was still observed in lymph nodes. PT had a strong mitogenic effect in vitro on lymph node cells but a weak effect on spleen cells indicating that the adjuvant activity of PT involves other effects besides the mitogenic activity.
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