Cytosine methylation and suppression of O6-methylguanine-DNA methyltransferase expression in human rhabdomyosarcoma cell lines and xenografts.
Human tumor cell lines that do not express O6-methylguanine-DNA methyltransferase (MGMT) in detectable quantities (Mer-) are hypersensitive to the effects of O6-guanine-alkylating agents. Because the Mer- phenotype enhances tumor response to such agents, we investigated possible mechanisms involved in regulation of MGMT expression in a panel of Mer+ and Mer- pediatric rhabdomyosarcoma xenograft and cell lines. All Mer- cell and xenograft lines lacked not only MGMT activity but also the protein and mRNA as well, suggesting that its expression is transcriptionally regulated. Transfection of Mer+ and Mer- rhabdomyosarcoma cell lines with MGMT gene promoter-CAT constructs yielded similar levels of CAT expression, indicating that Mer- cells possessed the necessary factors to support transcription. Methylation in the 5'-untranslated region of the MGMT gene was assayed by Southern analysis using methylation-sensitive restriction enzymes. Digestion with HpaII and its methylation-insensitive isoschizomer, MspI, revealed little overall correlation between methylation and MGMT expression. However, methylation in a single SmaI site at position-69 was observed in all MGMT deficient lines but not in any MGMT expressing lines. These results suggest that methylation of specific cytosines in the MGMT promoter may play a role in suppressing its expression, as well as being a potentially useful marker for the Mer- phenotype.
von Wronski
M A
MA
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Harris
L C
LC
Tano
K
K
Mitra
S
S
Bigner
D D
DD
Brent
T P
TP
D000818:Animals; D002460:Cell Line; D002648:Child; D003596:Cytosine; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007165:Immunosuppression Therapy; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D009368:Neoplasm Transplantation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012208:Rhabdomyosarcoma / Q000201:enzymology* / Q000235:genetics; D013489:Suppression, Genetic; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D003596:Cytosine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase