Gene amplification in Djungarian hamster cell lines possessing decreased plasma membrane permeability for colchicine and some other drugs

Abstract

By multistep selection a set of clones and sublines possessing different levels of resistance to colchicine or adriablastin was obtained from the SV40-transformed Djungarian hamster cell lines, DM-15 and DMcap. Resistance to both colchicine and adriablastin is associated with an alteration of plasma membrane permeability leading to a decreased uptake of various drugs (3H-colchicine, 3H-cytochalasin B, 3H-actinomycin D, 3H-puromycin, 3H-vinblastine, 14C-chloramphenicol). The DNA of cells highly resistant to cholchicine can transmit resistance only to low dosages of the drug. Comparison of DNAs from wild-type and resistant cells digested by restriction endonucleases revealed new classes of repeated DNA sequences in resistant cell lines. The degree of DNA repetition was correlated with the level of drug resistance. The repeated DNA sequences evidently represent parts of the genome that are amplified in resistant cells. The size of the amplified sequences is 200-250 kilobase pairs (kb). Cell lines highly resistant to colchicine contain amplified DNA, which like mitochondrial DNA replicate asynchronously with the main portion of the cellular DNA and related but not identical DNA sequences are amplified in independent cell lines selected for resistance to colchicine, adriablastin, and actinomycin D. These cell lines display similar patterns of alterations of plasma membrane permeability. The amplified DNA sequences may contain a gene or genes the overexpression of which leads to change in plasma membrane permeability and a development of resistance to various drugs.

Expression of phenotypic traits following modulation of colchicine resistance in J774.2 cells

“…The average number of DMs per cell displayed a close correlation with the level of colchicine resistance as has similarly been observed in vincristine-resistant MAZ mouse tumor cells (Meyers et al, 1985) and C-46 murine neuroblastoma cells (Baskin et al, 1981). DMs are indicative of gene amplification Schwab et al, 1983) and based on hybridization studies published by Roninson et al (1984) and Gudkov et al, (1985), multi-drug resistant cells contain amplified DNA sequences. Thus the decrease in the average number of DMs per cell suggests a decrease in the dosage of DNA sequences which encode resistance-specific proteins.…”

“…DMs are indicative of gene amplification Schwab et al, 1983) and based on hybridization studies published by Roninson et al (1984) and Gudkov et al, (1985), multi-drug resistant cells contain amplified DNA sequences. Thus the decrease in the average number of DMs per cell suggests a decrease in the dosage of DNA sequences which encode resistance-specific proteins.…”

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

“…6A), pMDR1 hybridized to mRNA in both human and mouse cell lines, whereas at higher stringency (Fig. 6B) (20), and Gudkov et al (14) suggested that the gene encoding multidrug resistance in Chinese hamster ovary (CHO) cells could be transferred from hamster to mouse L cells by DNA-mediated gene transfer and that this transfer correlates with increased expression of a 170-kilodalton membrane glycoprotein (8,20). However, the nature of the gene(s) transferred in these experiments has not yet been demonstrated at the DNA level.…”

“…This problem has been studied in tissue culture through the development of rodent and human cell lines which express simultaneous resistance to many drugs, such as colchicine, vinblastine, doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.), and actinomycin D (3,4,16). By use of the technique of in-gel renaturation (21), it has been possible to clone DNA sequences, designated mdr, that are commonly amplified in various hamster (13,14,22) and human (11,23,24) multidrug-resistant cell lines. The cloned human DNA sequence mdrl is expressed at high levels as a 4.5-kilobase (kb) mRNA in several different human multidrugresistant cell lines (23,24).…”

Multidrug Resistance of DNA-Mediated Transformants is Linked to Transfer of the Human mdr1 Gene

“…This problem has been studied in tissue culture through the development of rodent and human cell lines which express simultaneous resistance to many drugs, such as colchicine, vinblastine, doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.), and actinomycin D (3,4,16). By use of the technique of in-gel renaturation (21), it has been possible to clone DNA sequences, designated mdr, that are commonly amplified in various hamster (13,14,22) and human (11,23,24) multidrug-resistant cell lines. The cloned human DNA sequence mdrl is expressed at high levels as a 4.5kilobase (kb) mRNA in several different human multidrugresistant cell lines (23,24).…”