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Changes in O6-methylguanine-DNA methyltransferase expression during immortalization of cloned human fibroblasts
L C Harris1, M A von Wronski, C C Venable
1Department of Molecular Pharmacology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Abstract:
Suppressed expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), characterized as the Mer- phenotype, occurs only in malignant or transformed cell lines. To investigate the relationship between the transformation process and loss of MGMT expression, we derived 20 cloned lines of IMR90 normal fibroblasts transfected with the plasmid pSV3neo expressing the SV40 large-T antigen. Of the five lines that were grown until crisis phase, four emerged as continuously proliferating immortal lines. Of these, only one retained MGMT, the other three having become Mer-. In every case the loss of MGMT coincided with the final phase of immortalization following crisis. Because these were cloned cell lines it is clear that the phenotypic change to Mer- is not merely due to selection of a Mer- cell from the initial population, but must involve a cellular change in MGMT regulation. It is not clear if increased mutation rate associated with loss of MGMT results in increased frequency of an immortalization event or if an immortalization event, such as telomere disruption, results in MGMT suppression. In addition, we have shown that, consistent with previous observations, both hypermethylation in promoter sequences and hypomethylation of downstream sequences in the body of the gene were closely associated with loss of MGMT expression. These studies also illustrate the utility of these new cloned cell lines for characterizing molecular events associated with transformation and immortalization.
Insights
Loss of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) occurs during cell immortalization. This study shows MGMT suppression is a cellular change, not just selection, linked to epigenetic modifications during transformation.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme.
- Suppressed MGMT expression (Mer- phenotype) is observed in malignant and transformed cells.
- The relationship between cellular transformation and MGMT loss is not fully understood.
Purpose of the Study:
- To investigate the link between the transformation process and the loss of MGMT expression.
- To determine if MGMT suppression is due to selection or a cellular regulatory change.
- To characterize the epigenetic changes associated with MGMT loss during immortalization.
Main Methods:
- Transfection of normal IMR90 fibroblasts with SV40 large-T antigen plasmid.
- Derivation and culturing of 20 cloned cell lines.
- Monitoring cell proliferation, crisis phase, and immortalization.
- Analysis of MGMT expression and promoter/gene body methylation patterns.
Main Results:
- Four out of five immortalized cell lines lost MGMT expression (became Mer-).
- Loss of MGMT coincided with the final stages of immortalization post-crisis.
- MGMT suppression resulted from a cellular regulatory change, not selection.
- Hypermethylation of promoter regions and hypomethylation of downstream sequences correlated with MGMT loss.
Conclusions:
- Loss of MGMT expression is an active cellular event during fibroblast immortalization.
- Epigenetic modifications, including promoter hypermethylation, are key mechanisms for MGMT suppression.
- Cloned cell lines are valuable tools for studying molecular events in transformation and immortalization.