Amplified dihydrofolate reductase genes in unstably methotrexate-resistant cells are associated with double minute chromosomes.

Abstract

Karyotype Analysis. Midlogarithmic phase cultures were treated with 0.06 ,ug of Colcemid per ml and metaphase spreads were prepared by a modification of the procedure of Deaven and Petersen (10). Cells were gently trypsinized, centrifuged at 800 X g, and resuspended with 0.5 ml of residual medium; 5 ml of 75 mM KCI (pH 7.0) at 37°C was added by drops and the mixture was incubated for 5-15 min at 370C. Cells were again pelleted and resuspended in 0.5 ml of residual medium; 4 ml of ice-cold methanol/acetic acid, 3:1 (vol/vol), was added by drops and the mixture was placed on ice for 10 min. The cells were centrifuged again and fixation was repeated. Cells were finally centrifuged, resuspended in methanol/acetic acid, 3:1 (vol/vol), and dropped onto clean dry slides. Metaphases were stained for 10 min with 1 Mg of ethidium bromide per ml in 50 mM potassium phosphate (pH 6.8) or for 30 min with 2% Gurr's Giesma in 50 mM potassium phosphate (pH 6.8 KCI at 370C for 15 min. After pelleting, the cells were resuspended in 5 ml of 75 mM KCI, and 20 ml of 50% acetic acid was slowly added at room temperature. In parallel, metaphase spreads were prepared for determination of the mitotic index, which routinely exceeded 50%. Fixed cells were sheared by passage twice through a 23-gauge syringe needle with moderate pressure. An equal volume of 20% sucrose in 1 M hexylene glycol/0.5 mM 1,4-piperazinediethane sulfonic acid (Pipes), pH 6.8/0.1 mM CaCl2 was added and the suspension was passed through the 23-gauge syringe once more, centrifuged at 400 X g for 10 min to remove interphase nuclei, and then kept on ice. The supernatant, containing metaphase chromosomes, was layered onto a 1-liter linear sucrose gradient (20-50% sucrose in hexylene glycol/Pipes/CaCl2) and centrifuged (2500 rpm for 30 min at 200C) in a reorienting Sorvall SZ-14 Zonal Rotor. Fractions (100 ml) were collected and chromosomes were pelleted (14,500 X g for 10 min in a HB-4 Sorvall rotor), washed with 10 mM Tris.HCI, pH 8.0/10 mM NaCI/1 mM EDTA, recentrifuged, resuspended in 200-400 ,ul of Tris-HCl/NaCl/EDTA with 0.5% sodium dodecyl sulfate and 50 ,ug of proteinase K per ml (13)

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

“…The amplified genes may be located within expanded regions on extended single chromosomes (9,32) or in small, mitotically paired, chromosomal elements called double minutes (18,26). Metaphase spreads of parental S3 and MTX-resistant S3-300 cells indicated that the resistant line had acquired large numbers of double minute chromosomes (Fig.…”

Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

“…Cells grown in increasing concentrations of MTX developed resistance to the drug by overproducing its target enzyme, DHFR, as a result of amplification of the endogenous DHFR genes within the genome. In sta,ble transformants, the amplified DNA sequences usually reside in homogeneous staining regions of the chromosomes (11,34) some-like structures called double minutes (6,20). Subsequent studies showed that cloned DHFR genes and other genes cotransformed with them could also be amplified by this stepwise MTX selection when they were transferred into DHFR-deficient cells (10,21,22,32,35).…”

Human chromosome analysis in 24 cases of primary carcinoma of the large intestine: contribution of the G-banding technique

“…The genetic stuctures responsible for this have been identified as the homogeneously staining regions (HSR), related to a stable methotrexate resistance. Double-minutes confer variable resistance as a function of their quantitative importance (Alt et al, 1978;Bostock et al, 1979;Kaufman et at., 1979). In addition to their theoretical interest, these results open interesting perspectives on the possible relationships between the cytogenetic examination of a tumour and its chemo-sensitivity.…”

“…can be related to cancer progression and also to cytostatic drug resistance (Bostock et al, 1979;Kaufman et al, 1979). Cytogenetics could thus become a useful, perhaps even an essential tool in the classification of digestive cancers (Yunis et al, 1981).…”