Unbalanced growth in mouse cells with amplified dhfr genes

Abstract

When grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase (dhfr) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G1/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G1/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub-cytotoxic concentrations, also inhibits the cell cycle at the G1/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.

Trimethoprim: An Old Antibacterial Drug as a Template to Search for New Targets. Synthesis, Biological Activity and Molecular Modeling Study of Novel Trimethoprim Analogs

“…Folate metabolism has long been recognized as an important and attractive target for the development of therapeutic agents against bacterial, parasitic infections [1], and cancer therapy [2,3]. Dihydrofolate reductase (DHFR) is an essential enzyme, which catalyzes the reduction of dihydrofolate acid (7,8-dihydrofolate, DHF) to tetrahydrofolic acid (5,6,7,8-THF) using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor (Scheme 1) [4][5][6]. The crucial role of DHFR is related to biosynthesis pathways of the thymidylate and purines, as well as several other amino acids like glycine, methionine, serine, and N-formyl-methionyl tRNA [7][8][9].…”

Trimethoprim and other nonclassical antifolates an excellent template for searching modifications of dihydrofolate reductase enzyme inhibitors

“…Therefore, DHFR couples with thymidylate synthase (TS), which catalyzes the reductive methylation of deoxyuridine monophosphate in deoxythymidine monophosphate using N5 -N10-methylenetetrahydrofolate (5,10-Methylene THF) as a cofactor [165][166][167][168]. Despite the extensive targeting of DHFR in pathogenic organisms [153,[169][170][171], hDHFR is also targeted by anticancer agents [172][173][174][175]. MTX targets hDHFR and is a wellknown anticancer drug for the treatment of leukemia, breast cancer, lung cancer, osteosarcoma, and lymphoma [176].…”

In Silico Study Probes Potential Inhibitors of Human Dihydrofolate Reductase for Cancer Therapeutics

“…Dihydrofolate reductase (DHFR) is a ubiquitous enzyme and exists in a wide range of organisms [1]. DHFR is crucial for proper cellular growth and proliferation, where it regulates the maintenance of tetrahydrofolate (THF) and its derivatives, leading to the synthesis of purine and thymidylates [2]. DHFR is known for its enzymatic action to catalyze the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-THF.…”

“…Despite the extensive targeting of DHFR in pathogenic organisms [9,10,11,12], hDHFR is also targeted by anticancer agents [2,13,14,15]. Methotrexate (MTX) targets hDHFR and is a well-known anticancer drug for the treatment of leukemia, breast cancer, lung cancer, osteosarcoma, and lymphoma [16].…”