Topoisomerase II inhibition and altered kinetics of formation
and repair of nitrosourea and cisplatin--induced DNA interstrand cross-links and
cytotoxicity in human glioblastoma cells.
By altering the accessibility of DNA sequences for alkylation or platination,
and/or for subsequent repair, topoisomerase II can potentially affect the
level of DNA interstrand cross-links induced in cells by bifunctional agents.
In this study, we investigated the extent to which inhibition of topoisomerase
II activity in a human glioblastoma multiforme cell line alters the kinetics
of both the formation and the repair of total genomic DNA interstrand cross-links,
as well as the sensitivity of the tumor cells to cis- diamminedichloroplatinum
II (cis-DDP) and 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU). Cells were
incubated with and without 200 microM novobiocin, a known topoisomerase
II inhibitor, for 24 h, followed by exposure to 50 microM BCNU and 25 microM
cis-DDP. DNA interstrand cross- linking was determined at various time
points over 72 h, using a modified ethidium bromide-DNA binding assay.
Sensitivity of the cells to cis-DDP and BCNU was also determined with and
without novobiocin pretreatment with 200 microM novobiocin. This concentration
of novobiocin showed no significant direct cytotoxicity, although it inhibited
topoisomerase II activity in tumor cell nuclear extracts by 73%. A significant
decrease in the rate of repair of both cis-DDP and BCNU induced DNA interstrand
cross-links, with a corresponding decrease in the clonogenic survival of
the cells, was observed following novobiocin exposure. Although the peak
cross-link indices of novobiocin- treated cells relative to controls were
not significantly increased, residual DNA cross-linking in the cells after
72 h was increased by 1.4- fold for BCNU and 3-fold for cells treated with
cis-DDP, thus, indicating a greater effect of topoisomerase II on cross-link
repair than on cross-link formation. These data suggest that inhibition
of topoisomerase II may provide a potentially effective clinical strategy
for sensitizing human brain tumors, and possibly other tumors as well,
to DNA cross-linking anticancer agents.