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Determination of the DNA Content of a Six-Gene Amplicon in the Multidrug-resistant Chinese Hamster Cell Line CH^RB3 by Flow Karyotyping

Department of Molecular Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam [A. P. M. J., A. R.], and Laboratory for Radiobiology, University of Amsterdam, Academic Medical Centre, FO-212, Meibergdreef 9, 1105 AZ Amsterdam, [J. A. A.], The Netherlands

Acquired multidrug resistance in cultured cells is often due to amplification of pgp genes, which gives rise to overproduction of P-glycoproteins that confer resistance by reducing the intracellular drug accumulation. The size of these amplicons varies between multidrug resistant cell lines and is often much larger than the gene selected for. Amplicons of the multidrug resistant Chinese hamster ovary cell line CH^RC5 and its progenitor CH^RB3, for example, span at least five different genes besides the pgp genes. Linkage of these gene classes with pgp had been shown by in situ hybridization and by long distance mapping using pulsed field gradient gel electrophoresis. Because the boundaries of the larger amplicons could not be determined, the size of such amplicons is not yet known, even though the six genes span at least 1500 kilobases.

In the present study we have determined the amplicon size in B3⁺, a subclone of CH^RB3 with a homogeneously staining region on chromosome 7q+ that harbors the amplified genes. We estimated the amplicon size in revertant clones by correlating the decreased DNA content of the 7q+ homogeneously staining region with the number of lost amplicons. The reduction of the homogeneously staining region DNA that accompanied reversion was determined by flow cytometry of propidium iodide stained chromosome suspensions of the various cell lines. We found that about 107 megabase pairs were lost together with 11–24 P-glycoprotein gene copies, suggesting that the mean amplicon size is in the range of 4.5–10.1 megabase pairs.

¹ To whom requests for reprints should be addressed.

Received 9/18/89. Revised 12/27/89.