Phorbol esters rapidly attenuate glutamine uptake and growth in human colon carcinoma cells

Background. The amino acid glutamine, while essential for gut epithelial growth, has also been shown to stimulate colon carcinoma proliferation and diminish differentiation. Human colon carcinomas are known to extract and metabolize glutamine at rates several-fold greater than those of normal tissues, but the regulation of this response is unclear. Previously we reported that phorbol esters regulate hepatoma System ASC/B⁰-mediated glutamine uptake and cell growth. As human colon carcinoma cells use this same transporter for glutamine uptake, the present studies were undertaken to determine whether similar regulation functions in colon carcinoma. Materials and methods. Human colon carcinoma cell lines (WiDr and HT29) were treated with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and initial-rate transport of glutamine and other nutrients was measured at specific times thereafter. Growth rates were monitored during culture ± PMA or an excess of System ASC/B⁰ substrates relative to glutamine. Results. PMA treatment induced a rapid inhibition of glutamine uptake rates in WiDr and HT29 cells by 30 and 57%, respectively, after 1 h. Cycloheximide failed to block this response, indicating that the mechanism by which PMA exerts its effects is posttranslational. The inhibition of glutamine uptake by PMA was abrogated by the PKC inhibitor staurosporine, suggesting that this rapid System ASC/B⁰ regulation may be mediated by a PKC-dependent pathway. PMA also significantly decreased transport via System y⁺ (arginine) and System A (small zwitterionic amino acids). Chronic phorbol ester treatment inhibited WiDr cell growth, as did attenuation of System B⁰- mediated glutamine uptake with other transporter substrates. Conclusions. System ASC/B⁰ uptake governs glutamine-dependent growth in colon carcinoma cell lines, and is regulated by a phorbol ester-sensitive pathway that may involve PKC. The results further establish the link between glutamine uptake and colon carcinoma cell growth, a relationship worthy of further investigation with the goal of discovering novel cancer therapeutic targets. (C) 2000 Academic Press.