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Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea

Snapka, Robert M. and Varshavsky, Alexander (1983) Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea. Proceedings of the National Academy of Sciences of the United States of America, 80 (24). pp. 7533-7537. ISSN 0027-8424. PMCID PMC389986. doi:10.1073/pnas.80.24.7533.

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Previous work has shown that mammalian cells that carry unstably amplified genes for dihydrofolate reductase (DHFR) gradually lose the amplified DHFR genes when grown in the absence of the DHFR inhibitor methotrexate (MTX). Unstably amplified genes occur on small acentric chromosomes called double minutes (DMs) or even smaller chromatin fragments, in contrast to stably amplified genes, which reside in centromere-containing chromosomes. We have found that the rate of loss of the unstably amplified DHFR genes can be greatly increased by growing the cells in the presence of a nonlethal concentration of hydroxyurea. For example, in one MTX-resistant subline studied, approximate to 90% of the original DHFR gene dosage is lost in 25-30 cell doublings in the absence of MTX. The same degree of loss is achieved, however, in <4 doublings if cells are grown in the presence of 50 μM hydroxyurea. This new effect of hydroxyurea does not appear to be due to changes in plating efficiency or selective cytotoxicity. In particular, no increase in cell death occurs at 50 μM hydroxyurea, and cells continue to multiply, albeit 1/2 to 2/3 as fast as in the absence of hydroxyurea. The ability to selectively accelerate the loss of amplified genes from mammalian cells as shown in the present work may have important implications both for the problem of drug resistance in cancer chemotherapy and for curing mammalian cells of extrachromosomally maintained DNA genomes of pathogenic viruses.

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URLURL TypeDescription CentralArticle
Varshavsky, Alexander0000-0002-4011-258X
Additional Information:© 1983 by the National Academy of Sciences. Communicated by Howard Green, August 29, 1983. We are greatly indebted to Robert Schimke, Randall Kaufman, and James Barsoum for the pDHFR11 cDNA clone and R500 cells; to Vladimir Volloch for the pCRI-M9 cDNA clone; and to James Barsoum, Igor Roninson, and Paul Swerdlow for their comments on the manuscript. This work was supported by grants to A.V. from the National Cancer Institute (CA30367 and CA33297). R. M.S. was supported by a fellowship from the Flow General Royalty Fund. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Funding AgencyGrant Number
Flow General Royalty FundUNSPECIFIED
Subject Keywords:gene amplification and loss, "curing" mammalian cells of acentric chromosomal fragments
Issue or Number:24
PubMed Central ID:PMC389986
Record Number:CaltechAUTHORS:SNApnas83
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1021
Deposited By: Tony Diaz
Deposited On:01 Dec 2005
Last Modified:08 Nov 2021 19:06

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