Targeting of O6-methylguanine-DNA methyltransferase (MGMT) activity by antimessenger oligonucleotide sensitizes CHO/Mex+ transfected cells to mitozolomide.
The targeting of mRNA with antisense oligonucleotides is increasingly employed to inhibit the expression of gene function. Since the level of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is decisive in protection of cells against damage produced by alkylating agents, including cytostatic drugs, the targeted inhibition of this repair activity might be of importance for therapeutic approaches. In order to investigate whether antisense targeted MGMT depletion is feasible to transiently modify the sensitivity of cells to anticancer drugs, we studied the expression of MGMT and cellular sensitivity upon inhibitor and antisense treatment using CHO transfectants expressing human MGMT. It was shown by polymerase chain reaction that antisense oligonucleotides specifically inhibited MGMT mRNA level. Nevertheless, MGMT protein was found not to be reduced significantly, as demonstrated by Western blotting. Correspondingly, no significant decrease in MGMT activity was observed, as measured 36 h after MGMT antisense oligonucleotide administration. Given together with the MGMT depleting agent O6-methylguanine, reduction in MGMT protein as well as activity was found. MGMT antisense oligonucleotide enhanced the sensitivity of cells to the tumor therapeutic drug mitozolomide, as measured by sister chromatid exchange formation. This sensitization was further enhanced by combined treatment with antisense oligonucleotide and O6-methylguanine, indicating that MGMT antisense can be supportive in sensitization of cells to an alkylating drug.
Citti
L
L
Istituto di Mutagenesi e Differenziamento CNR, Pisa, Italy.
Boldrini
L
L
Preuss
I
I
Kaina
B
B
Mariani
L
L
Rainaldi
G
G
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D018390:Gene Targeting; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology*; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D012854:Sister Chromatid Exchange / Q000187:drug effects; D014162:Transfection
D000970:Antineoplastic Agents; D009588:Nitrogen Mustard Compounds; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; C040369:mitozolomide; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase