CHINESE-HAMSTER OVARY CELLS RESISTANT TO THE TOPOISOMERASE-II CATALYTIC INHIBITOR ICRF-159 - A TYR49PHE MUTATION CONFERS HIGH-LEVEL RESISTANCE TO BISDIOXOPIPERAZINES
M. Sehested et al., CHINESE-HAMSTER OVARY CELLS RESISTANT TO THE TOPOISOMERASE-II CATALYTIC INHIBITOR ICRF-159 - A TYR49PHE MUTATION CONFERS HIGH-LEVEL RESISTANCE TO BISDIOXOPIPERAZINES, Cancer research, 58(7), 1998, pp. 1460-1468
Anticancer drugs targeted to the nuclear enzyme DNA topoisomerase II a
re classified as poisons that lead to DNA breaks of catalytic inhibito
rs that appear to completely block enzyme activity, To examine the eff
ects of the bisdioxopiperazine class of catalytic inhibitors to topois
omerase II, we investigated a Chinese hamster ovary (CHO) subline sele
cted for resistance to ICRF-159 (CHO/159-1). Topoisomerase II alpha co
ntent in CHO/159-1 cells was reduced by 40-50%, compared to wild-type
CHO cells, whereas the beta isoform was increased by 10-20% in CHO/159
-1 cells. However, the catalytic activity of topoisomerase II in nucle
ar extracts from CHO/159-1 cells was unchanged, as was its inhibition
by the topoisomerase II poison etoposide (VP-16). No inhibition of top
oisomerase II catalytic activity by ICRF-187 was seen in CHO/159-1 cel
ls up to 500 mu M, whereas inhibition was evident at 50 mu M in wild-t
ype CHO cells, VP-16-mediated DNA single-strand breaks and cytotoxicit
y were similar in the two sublines, ICRF-187 could abrogate these VP-1
6 effects in the wild-type line but had no effect in CHO/159-1 cells,
Western blots of topoisomerase II alpha after incubation of CHO cells
with ICRF-187 demonstrated a marked band depletion, whereas this effec
t was completely lacking in CHO/159-1 cells, and an equal effect of VP
-16 was observed in both lines, These data imply that the CHO/159-1 to
poisomerase II alpha lacks sensitivity to bisdioxopiperazines and that
the mechanism of resistance in this cell line does nor confer cross-r
esistance to topoisomerase II poisons, suggesting that mutations confe
rring resistance to bisdioxopiperazines can occur at sites distinct fr
om those responsible ibr resistance to complex stabilizing agents, Acc
ordingly, CHO/159-1 cDNA showed two heterozygous mutations in the prox
imal NH2-terminal part of topoisomerase II alpha (Tyr49Phe and Delta 3
09Gln-Gln-Ile-Ser-Phe313), which is in contrast to those induced by to
poisomerase II poisons, which cluster further downstream. Site-directe
d mutagenesis and transformation of the homologous Tyr50Phe coding mut
ation in human topoisomerase II alpha in a temperature-conditional yea
st system demonstrated a high-level resistance to ICRF-193, compared t
o cells expressing wild-type cDNA, but none toward the poisons VP-16 o
r amsacrine, thus confirming that the Tyr50Phe mutation confers specif
ic resistance to bisdioxopiperazines. Thus, these results indicate tha
t the region of the protein involved in ATP-binding also plays a criti
cal role in sensitivity to bisdioxopiperazines, a result consistent wi
th the known requirement for the formation of an ATP-bound closed clam
p for bisdioxopiperazine activity. These results may enable a more pre
cise understanding of the interaction of topoisomerase II-directed dru
gs with their target enzyme.