Differential Alterations of Dihydrofolate Reductase Gene in Human Leukemia Cell Lines Made Resistant to Various Folate Analogues

In order to clarify a molecular mechanism of folate resistance in leukemia cells, we studied alterations of the dihydrofolate reductase (DHFR) gene in a human leukemia cell line, MOLT‐3, and its sublines made resistant to methotrexate (MTX), trimetrexate (TMQ) and N(10)‐propargyl‐5,8‐dideazafolic acid (CB3717), alone or in combination. Major alterations of the DHFR gene were examined by Southern analysis of high‐molecular‐weight DNA. The presence of a base change (T→C) at nucleotide position 91 of the DHFR gene, which is reported to be responsible for the reduced affinity of the enzyme for MTX in an MTX‐resistant human colon carcinoma cell, was examined by allele‐specific oligonucleotide hybridization. In a 10,000‐fold MTX‐resistant subline (MOLT‐3/MTX(10,000)), the normal allele of DHFR gene had been amplified. In contrast, a 200‐fold TMQ‐resistant subline (MOLT‐3/TMQ(200)) and a 30‐fold CB3717‐resistant subline selected from MOLT‐3/TMQ(200) (MOLT‐3/TMQ(200)‐CB‐3717(30)) were shown to have the mutant allele. Furthermore, the mutant allele had been amplified in a 500‐fold MTX‐resistant subline, which was established by the continuous exposure of the MOLT‐3/TMQ(200) cells to stepwise increases of drug concentration and designated as MOLT‐3/TMQ(200)‐MTX(500). On the other hand, a 40‐fold‐resistant subline to CB3717 alone (MOLT‐3/CB3717(40)) showed the normal allele without amplification. These data suggest that complex alterations of the DHFR gene are involved in the molecular mechanisms of folate resistance that can be differentially introduced into leukemia cells by exposure to various folate analogues, alone or in combination.