Dl. Kramer et al., LYSOSOMAL SEQUESTRATION OF POLYAMINE ANALOGS IN CHINESE-HAMSTER OVARYCELLS RESISTANT TO THE S-ADENOSYLMETHIONINE DECARBOXYLASE INHIBITOR, CGP-48664, Cancer research, 58(17), 1998, pp. 3883-3890
CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-adenosy
lmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1
clinical trials as an experimental anticancer agent. We have shown pre
viously (D. L. Kramer et at, J. Biol, Chem,, 270: 2124-2132, 1995) tha
t Chinese hamster ovary (CHO) cells that are made resistant to the gro
wth inhibitory effects of the drug overexpress AdoMetDC because of a s
table gene amplification. Unexpectedly, these same cells (CHO/644) wer
e found to be insensitive to the growth inhibitory effects of N-1-N-11
-diethylnorspermine (DENSPM)-a polyamine analogue also undergoing Phas
e 1 clinical trials-despite accumulating similar to 5 times more analo
gue than parental cells. We now report that treatment of CHO/664 cells
with DENSPM results in the formation of numerous large cytoplasmic va
cuoles, which on the basis of electron microscopy and cytochemical sta
ining seem to be lysosomal in origin. A series of newly established CH
O cell lines made differentially resistant to 1, 3, 10, 30, and 100 mu
M CGP-48664 by chronic exposure were used to demonstrate that vacuole
formation correlated with the accumulation of extremely high levels o
f DENSPM without increasing growth inhibition. These same cells were u
sed to show that AdoMetDC gene overexpression as indicated by mRNA lev
els was unrelated to vacuole formation; cells resistant to 100 mu M CG
P-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and form
ed vacuoles in response to DENSPM, whereas those resistant to 10 mu M
CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and fa
iled to form vacuoles, Despite accumulating to high intracellular leve
ls, DENSPM was much Less effective than spermine at down-regulating or
nithine decarboxylase and polyamine transport activities in highly res
istant cells. Similarly, DENSPM was less able to induce spermidine/spe
rmine N-1-acetyltransferase activity in cells that formed vacuoles tha
n in those that did not. Overall, natural polyamines failed to induce
vacuoles and various analogues of DENSPM were used to probe the struct
ural specificity of the effect. The data are consistent with the proba
bility that DENSPM is sequestered to high concentrations in lysosomal
vacuoles of CGP-48664-resistant cells and is, therefore, not available
to interact with polyamine regulatory sites or to cytotoxically affec
t cell growth. In addition to implicating the lysosome as a potential
new site of CGP-48664 drug action that could be involved in antitumor
activity and/or host toxicities, the findings also suggest a potential
mechanism of cell resistance to analogues such as DENSPM.