M. Kamishohara et al., Selective accumulation of the endoplasmic reticulum-Golgi intermediate compartment induced by the antitumor drug KRN5500, EXP CELL RE, 256(2), 2000, pp. 468-479
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.