Ceramide glycanase activities in human cancer cells

Ceramide glycanase (CGase) activities have been detected in different human tumor cells (colon, carcinoma Colo-205; neuroblastoma, IMR-32; breast canc er lines, SKBr3 and MCF7). However, the level of enzymatic activity is lowe r in these cells compared to that present in other mammalian tissues report ed before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S. Metho ds Enzymol. (in press)). The majority of CGase activity was found in the 10 0,000g soluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble enzyme, the requirement for optimum CGase activi ty was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998) Acta Pol. Biochim. 42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a protein to detergent ratio of one. All the mammalian CGase s, including human cancer cells, show an optimum pH between 5.5 and 5.8 in sodium acetate buffer. The CGase activities from cancer cells are found to be cation-independent; however, mercury, zinc, and copper ions seem to inhi bit the enzyme activity substantially in both tumor cells lines. The mercur y ion inhibition of CGase activities from ail different sources indicates a possible structural homology in the CGase proteins. Radiolabeled substrates, labeled at the sphingosine double bond or at the 3 -position of sphingosine without modifying double bond of sphingosine were used in this investigation. Both were active substrates with all enzyme pre parations isolated from different cancer cells (apparent Km, 500 mu M for n LcOse5[H-3-DT]Cer and 350 mu M for GgOse4[sph-3-H-3]Cer with Colo-205 enzym e). Structural analogues of ceramide and sphingosine (L-PPMP, L-PDMP, alkyl amines, and Tamoxifen) inhibited cancer cell CGase activities in vitro.