Characterisation of methotrexate-resistant clones

Three methotrexate (MTX)-resistant clones M1, M2 and M3 have been isolated from Chinese hamster V79 cells and characterised for aneuploidy, chromosomal aberrations, sister-chromatid exchange (SCE) mutation and transfection. Amplification of the dihydrofolate reductase (DHFR) gene in these clones has been established from (a) direct measurement of DHFR activity, (b) existence of double minute chromosomes and (c) homogeneously staining region (HSR) in chromosome number 2 by G-banding technique. Clone M1 on further exposure to gradually increasing concentrations of MTX gave rise to two more clones M4 and M5, resistant to 1200 nM and 2400 nM MTX, respectively. The levels of folate reductase activity in clones M4 and M5 were 21.90 units per 10(7) cells and 33.30 units per 10(7) cells, whereas the value was 13.90 units per 10(7) cells in clone M1 and 1.1 units per 10(7) cells in normal V79 cells. Increased chromosomal aberrations were observed in each of these clones in comparison to those in normal V79 cells. There was a correlation between the increase in DHFR activity and the increase in the level of background SCE as well as the increase in aneuploidy. However, background mutation frequency at the HGPRT locus remained unaffected though transfection frequency decreased.