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Interruption of base excision repair selectively enhances
temozolamide and 1,3-bis(2-chloroethyl)-nitrosurea cytotoxicity in treatment of
adult human malignant glioma cell lines
Meg Verrees, Lili Liu, Stanton L. Gerson
University Hospitals of
Cleveland/Case Comprehensive Cancer Center, Cleveland, OH. E-mail: megverrees@yahoo.com
Introduction. Effective base excision repair (BER) confers resistance on tumor
cells through reversal of damage inflicted alkylating chemotherapeutic agents.
Temozolomide (TMZ) and 1,3-bis(2-chloroethyl)-nitrosurea (BCNU) produce their
deleterious effects via attachment of bulky adducts to the DNA backbone or
through formation of DNA interstrand crosslinks.
The BER pathway intrinsically
reverses damage by removal of altered bases, leaving exposed
apurinic/apyrimidinic (AP) sites.
Specific glycosylases instigate formation of
the AP site, causing a kink in the backbone of DNA.
AP endonuclease (APE) fits
into the bend at the newly-formed AP site and cuts the DNA backbone, thus
relieving the kink.
DNA polymerase-beta then fills the gap with the proper base
and DNA ligase fills the nick in the DNA backbone, respectively restoring
genomic fidelity and structural integrity of DNA.
Methoxyamine (MX) is a novel compound that inhibits BER by competing with APE
for the AP site and preventing continuation of repair at this early juncture in
the pathway.
Methods. Glioblastoma cell lines SWB 33, SWB 39, SWB 40, SWB 61 and LN 18
were expanded on multi-well plates, exposed to TMZ or BCNU and incubated for 9
days with or without addition of MX.
The plates were then analyzed via MTT
assays.
Five million cells of the SWB 40 and SWB 39 cell lines were injected into the
bilateral flanks of athymic mice.
Three weeks following inoculation, mice were
treated with either TMZ, TMZ and MX or MX alone (control).
Tumors were measured
three times per week.
Western blot analyses were used to elucidate the presence of BER proteins.
Results. Significant increase in TMZ cytotoxicity was noted in SWB 40 in both
MTT and nude mouse studies, with 2-fold increased sensitivity with MX at 10 uM
TMZ in the in-vitro studies and tumor growth delay and tumor doubling time of
> 21 days in-vivo.
LN 18 demonstrated a > 2-fold increased sensitivity to TMZ and BCNU in
presence of MX in in-vitro assays.
SWB 33 exhibited a 2-fold increased sensitivity in presence of MX at 45 uM
TMZ.
SWB 39 and 61 demonstrated no potentiation by MX of TMZ toxicity in in-vitro
studies.
Similarly, SWB 39 exhibited no potentiation in nude mouse assays.
All 5 cell lines demonstrated strong presence of BER proteins APE and DNA
polymerase-beta, however only SWB 40 and LN 18--those cell lines most sensitized
by MX--showed presence of presence of BER protein XRCC1.
Conclusions. MX interrupts BER and sensitized 3 of 5 malignant glioma cell
lines to TMZ and BCNU.
Sensitization may be linked to expression of XRCC1.
Select glioma cell lines may be targets for combined MX and TMZ or MX and
BCNU therapy.
Future directions. 1. To investigate other adult gliomas and pediatric brain
tumors and include orthotopic xenograft analyses.
2. To characterize the role of XRCC1 in glioma sensitization.
3. To evaluate dose response.
Copyright © 2004 American Association for Cancer Research. All rights
reserved.
Source: http://aacr04.agora.com/planner/displayabstract.asp?presentationid=4262
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