Comparison of O-6-methylguanine DNA methyltransferase (MGMT) mRNA levels in Mer+ and Mer- human tumor cell lines containing the MGMT gene by the polymerase chain reaction technique.
O-6-Alkylguanine is a mutagenic and carcinogenic DNA lesion induced by a variety of alkylating agents, including the chloroethylnitrosoureas. The lesion is repaired by the alkyl-accepting suicide enzyme O-6-methylguanine DNA methyltransferase (MGMT). Approximately 25% of cell lines derived from human tumors are phenotypically deficient in this enzyme and are described as Mer-. Recent cloning of the human MGMT cDNA (Tano, K.; Shiota, S.; Collier, J.; Foote, R.S.; Mitra, S. Proc. Natl. Acad. Sci. USA 87:686-690; 1990) has allowed for a more detailed analysis of the basis of the Mer- phenotype in human Mer- tumor cell lines. Using the polymerase chain reaction (PCR) technique, an MGMT cDNA probe based on the published sequence was generated. The probe and the PCR technique were then used to analyze the presence and expression of the MGMT gene in two Mer+ and four Mer- lines, including one SV40-transformed Mer- line and three Mer- human tumor cell lines. The data demonstrate that while all six cell lines contained a relatively nonamplified, nonrearranged MGMT gene, Mer- lines contained levels of MGMT mRNA detectable only by PCR analysis. Of the three Mer- tumor cell lines examined, two (COLO 320 HSR, A1235) contained MGMT mRNA levels that were four to five orders of magnitude lower than that of the prototype Mer+ tumor line (HT-29), while one (BE) contained no consistently detectable MGMT mRNA. These results suggest that in the human Mer- tumor lines tested, the Mer- phenotype was mediated by a severe reduction in MGMT mRNA levels, despite the presence of the MGMT gene.
Pieper
R O
RO
Section of Hematology and Oncology, Stritch School of Medicine, Loyola University Medical Center, Maywood, IL 60153.
Futscher
B W
BW
Dong
Q
Q
Ellis
T M
TM
Erickson
L C
LC
D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000235:genetics; D015870:Gene Expression; D006657:Histones / Q000235:genetics; D006801:Humans; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D015183:Restriction Mapping; D014407:Tumor Cells, Cultured / Q000201:enzymology*
D004273:DNA, Neoplasm; D006657:Histones; D012333:RNA, Messenger; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase