Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide.
BACKGROUND
The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins.


METHODS
Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing.


RESULTS
The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%).


CONCLUSIONS
The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell lines.
Department of Radiation Oncology, VU University Medical Center, P.O Box 7057, Amsterdam 1007 MB, The Netherlands. ka.vannifterik@vumc.nl
K A
KA
van Nifterik
J
J
van den Berg
W F
WF
van der Meide
N
N
Ameziane
L E
LE
Wedekind
R D M
RD
Steenbergen
S
S
Leenstra
M V M
MV
Lafleur
B J
BJ
Slotman
L J A
LJ
Stalpers
P
P
Sminia
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D018899:CpG Islands; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005909:Glioblastoma / Q000188:drug therapy* / Q000473:pathology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D011401:Promoter Regions, Genetic; D000077204:Temozolomide; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D018906:Antineoplastic Agents, Alkylating; D025521:Tumor Suppressor Proteins; D003606:Dacarbazine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; D000077204:Temozolomide