This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wani, M. A. Articles by Stambrook, P. J. Search for Related Content PubMed PubMed Citation Articles by Wani, M. A. Articles by Stambrook, P. J.

Increased Methotrexate Resistance and dhfr Gene Amplification as a Consequence of Induced Ha-ras Expression in NIH 3T3 Cells¹

Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521

Oncogene activation and loss of tumor suppressor genes are known to play a role in tumor initiation as well as its progression. The potential roles of these genes in perturbation of genome stability has become of major interest. To better understand the relationship between expression of an oncogene and genetic instability, we have studied a cell line expressing an activated human Ha-ras under the control of bacterial lactose operon regulatory elements for changes in methotrexate resistance and dihydrofolate reductase (dhfr) gene amplification following mutant Ha-ras induction. In these cells mutant Ha-ras is directed by an inducible SV40 promoter containing a bacterial lac operator sequence which is repressed due to constitutive expression of bacterial lac repressor gene. The expression of this Ha-ras is specifically induced by the addition of isopropyl-1-thio-ß-D-galactopyranoside (IPTG), a lactose analogue, to the culture medium. During single-step methotrexate selection, these cells showed an increased frequency of methotrexate resistance in the presence of IPTG. More than 60% of the methotrexate-resistant colonies showed a 2–6-fold amplification of the dhfr gene. One clone with rearranged dhfr had about 100-fold amplification of the gene. The increased capacity to amplify DNA in response to mutant Ha-ras induction was not locus specific since cells also displayed an increased frequency of resistance to N-(phosphonacetyl)-L-aspartic acid in the presence of ITPG. Four of the methotrexate-resistant clones with amplified dhfr gene were cultured further in the presence or absence of IPTG and subsequently compared for their ability to grow in soft agar as a measure of transformation. In medium containing methotrexate but no IPTG, the clones were unable to grow in soft agar, indicating that methotrexate resistance due to gene amplification is separable from transformation.

¹ This work was supported by NIH Grants P01 ES05652 and P30 ES06096. M. A. W. was supported by Training Grant ES07250 from NIH.

² To whom requests for reprints should be addressed, at Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, OH 45267-0521.

Received 10/18/93. Accepted 2/23/94.

This article has been cited by other articles:

				X. Chen, N. Mitsutake, K. LaPerle, N. Akeno, P. Zanzonico, V. A. Longo, S. Mitsutake, E. T. Kimura, H. Geiger, E. Santos, et al. Endogenous expression of HrasG12V induces developmental defects and neoplasms with copy number imbalances of the oncogene PNAS, May 12, 2009; 106(19): 7979 - 7984. [Abstract] [Full Text] [PDF]

				J. A. Knauf, B. Ouyang, E. S. Knudsen, K. Fukasawa, G. Babcock, and J. A. Fagin Oncogenic RAS Induces Accelerated Transition through G2/M and Promotes Defects in the G2 DNA Damage and Mitotic Spindle Checkpoints J. Biol. Chem., February 17, 2006; 281(7): 3800 - 3809. [Abstract] [Full Text] [PDF]

				L. S. Agapova, J. L. Volodina, P. M. Chumakov, and B. P. Kopnin Activation of Ras-Ral Pathway Attenuates p53-independent DNA Damage G2 Checkpoint J. Biol. Chem., August 27, 2004; 279(35): 36382 - 36389. [Abstract] [Full Text] [PDF]

				J. A. Fagin Minireview: Branded from the Start--Distinct Oncogenic Initiating Events May Determine Tumor Fate in the Thyroid Mol. Endocrinol., May 1, 2002; 16(5): 903 - 911. [Abstract] [Full Text] [PDF]

				H. I. Saavedra, K. Fukasawa, C. W. Conn, and P. J. Stambrook MAPK Mediates RAS-induced Chromosome Instability J. Biol. Chem., December 31, 1999; 274(53): 38083 - 38090. [Abstract] [Full Text] [PDF]

				L. H. Hartwell, P. Szankasi, C. J. Roberts, A. W. Murray, and S. H. Friend Integrating Genetic Approaches into the Discovery of Anticancer Drugs Science, November 7, 1997; 278(5340): 1064 - 1068. [Abstract] [Full Text]