Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Abstract

Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 μM MTX (in wild type the LD99.9 is 0.2 μM). MTX(R) phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.

Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization

“…The enzyme has been extracted from seedling of pea (Suzuki and Iway 1970), soybean (Reddy and Rao 1976), corn (Crosti 1981) and from suspension cultured cells of carrot and rice (Celia et aL, 1983), corn (Shimamoto and Nelson 1981), petunia (Barg et aL, 1984).…”

“…Selection of cell lines resistant to such antifolic agents as aminopterin and methotrexate (Mtx) has been reported (Barg et aL, 1984;Cella et aL, 1984;Mastrangelo and Smith 1977;White 1982). In some instances it was shown that these lines had an increased content of DHFR but dhfr gene amplification has not yet been demonstrated in plants.…”

Methotrexate Resistance in Datura innoxia (Uptake and Metabolism of Methotrexate in Wild-Type and Resistant Cell Lines)

“…One system has high affinity for MTX-free medium (data not shown). This is in contrast with the selected MTX-resistant cell lines of petunia (Barg et al, 1984) and carrot (Lazar et al, 1989), which were more unstable. Creason and Gonzales (1988) reported that uptake of MTX into tobacco cells is saturable, with a K, of 50 PM and a V,,, of 1 nmol h-'g-' fresh weight.…”

Elevated amounts of methotrexate-binding protein, different from normal dihydrofolate reductase, in a petunia MTXR-cell line

“…Mutants resistant to MTX or aminopterin have been isolated in culture of several plant species (5,8,22,24,28,29,32). In a previous paper we reported the isolation, upon a stepwise selection procedure, of several petunia cell lines resistant to MTX (5).…”

“…In a previous paper we reported the isolation, upon a stepwise selection procedure, of several petunia cell lines resistant to MTX (5). Preliminary biochemical examination of their mode of resistance showed that, compared with their wild type parental cell-line (WT), all mutants have elevated 3H-MTX-binding capacity and e nhanced DHFR activity as exhibited after gel electrophoresis.…”