Carcinogenesis, Vol. 20, No. 12, 2361-2365,
December 1999
© 1999 Oxford University Press
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O6-Methylguanine-DNA methyltransferase activity, p53 gene status and BCNU resistance in mouse astrocytes
1 Department of Oncology,
2 Department of Pathology and
3 Department of Clinical Neurological Sciences, University of Western Ontario and
4 London Regional Cancer Centre, London, Ontario N6A 4L6, Canada and
5 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S.Hershey Medical Center, Hershey, PA 17033, USA
We observed previously that wild-type p53 rendered neonatal mouse astrocytes resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a gene dose-dependent fashion. This effect of p53 appeared to be unrelated to its cell cycle regulation or apoptotic functions. Because in many cell types O6-methylguanine-DNA methyltransferase (MGMT)-mediated DNA repair is an important mechanism of resistance to nitrosoureas, we measured MGMT activity in wild-type, heterozygous and p53 knockout neonatal mouse astrocytes. Wild-type p53 astrocytes had significantly greater MGMT activity than either heterozygous or p53 knockout astrocytes: MGMT activity was ~5-fold greater in wild-type p53 astrocytes than in p53 knockout cells. However, despite successful depletion of MGMT activity in wild-type astrocytes by O6-benzylguanine (BG), resistance to BCNU persisted unchanged. Moreover, we excluded the possibility that continued resistance to BCNU at the concentrations used could be explained by a compensatory induction of MGMT triggered by exposure to either BCNU or BG. Although these studies support a role for p53 regulation of MGMT in neonatal mouse astrocytes, BCNU resistance in wild-type cells appears to be mediated by a non-MGMT mechanism. Nevertheless, regulation of DNA repair by MGMT may be another mechanism by which alterations of the p53 gene promote tumor initiation or progression.
Abbreviations: +/+, wild-type; +/–, heterozygous; –/–, knockout; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BG, O6-benzylguanine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MGMT, O6-methylguanine-DNA methyltransferase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline.
6 Present address: Section of Neurobiology, Yale University School of Medicine, New Haven, CT 06510, USA
7 To whom correspondence should be addressed at: London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario N6A 4L6, Canada Email: gcairncross{at}lrcc.on.ca
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