Selective accumulation of the endoplasmic reticulum-Golgi intermediate compartment induced by the antitumor drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C-14 unsaturated fatty acid through glycine and to the amino group of adenine, The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferati ve effect of KRN5500 remains to be elucidated, We have found that acute exp osure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug result s initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells character ized by numerous intracellular vacuoles, These KRN5500-resistant tumor cell s exhibited increased staining with the Golgi stain NBD C-6-ceramide and th e ER-Golgi fluorescent dye BODIPY-brefeldin A, which, unlike the parental d rug-sensitive cells, was dispersed throughout the cytoplasm, Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc trans ferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resi stant cell lines while the trans-Golgi marker enzyme, galactosyltransferase , was not, The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-S3, a cis-Golgi marker protein of the ER Golgi i ntermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtu bule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determin ant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstra te that KRN5500 alters the distribution of newly synthesized carcinoembryon ic antigen within the secretory pathway, including arrest of this N-glycosy lated protein in the Golgi of LS-174T colon carcinoma cells.