Abstract
Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF), an anti-purine agent that requires conversion to polyglutamate forms to be a potent inhibitor of its target enzymes. The sensitivity of cells continuously exposed to DATHF was not influenced by FPGS activity. After short term exposure to DATHF, cells expressing higher levels of human FPGS were more sensitive to the drug while cells expressing low levels were resistant. Cells expressing E. coli FPGS solely in the cytosol were resistant to DATHF and accumulated only low levels of the drug. Expression of E. coli FPGS in the mitochondria of these cells restored drug sensitivity and cytosolic drug accumulation. DATHF is extensively metabolized in the mitochondria of mammalian cells and, despite an inability to enter the mitochondria, folylpolyglutamates and DATHF polyglutamates formed in the mitochondria are released into the cytosol without prior hydrolysis. DATHF was not solely an inhibitor of de novo purine biosynthesis but also inhibited glycine synthesis in the cell.
Highlights
Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF’),an anti-purine agent that requires conversion to polyglutamate formsto be a potent inhibitorof its target enzymes.The sensitivity of cells continuouslyexposed to DATHF wasnotinfluenced byFPGS activity
The abbreviations used are: FPGS, folylpoly-y-glutamate syntheFor pulse treatment with DATHF, cells were plated in duplicate at a tase; CHO, Chinese hamster ovary; DMEM, deficient a-minimal essential medium lacking purines, thymidine (T), glycine (G), and folate; H, density of 2 x lo4 per 35-mm plate with 2 ml of the same medium and hypoxanthine; FBS, fetal bovine serum; dFBS, dialyzed fetal bovine allowed to attach to the surface of the plate for about 4 h,and the original medium was replaced by medium containing different levels of serum; AICAR, aminoimidazole ribonucleotide; PBS, phosphate-buffered saline; ReGlu, pteroylglutamic acid, folic acid; H,PteGlu, tetra- DATHF
Was used as a n example of a class of anti-folate agents that target de novo purine biosynthesis and which are considered to be pro-drugs in that theirpolyglutamate forms are much more potent inhibitors of target enzymes than are the parentmonoglutamate forms. Many of these compounds behave similarlyto reduced folates. They are effectively transported by mammalian cells, and they are good substrates for mammalian FPGS
Summary
1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Chinese hamster ovary (CHO) cells expressing human and Escherichia coli folylpolyglutamate synthetase (FPGS) activities were used as models to study factors regulating the cytotoxicity and metabolism of 5-deazaacyclotetrahydrofolate (DATHF’),an anti-purine agent that requires conversion to polyglutamate formsto be a potent inhibitorof its target enzymes.The sensitivity of cells continuouslyexposed to DATHF wasnotinfluenced byFPGS activity. The abbreviations used are: FPGS, folylpoly-y-glutamate syntheFor pulse treatment with DATHF, cells were plated in duplicate at a tase; CHO, Chinese hamster ovary; DMEM, deficient a-minimal essential medium lacking purines, thymidine (T), glycine (G), and folate; H, density of 2 x lo per 35-mm plate with 2 ml of the same medium and hypoxanthine; FBS, fetal bovine serum; dFBS, dialyzed fetal bovine allowed to attach to the surface of the plate for about 4 h,and the original medium was replaced by medium containing different levels of serum; AICAR, aminoimidazole ribonucleotide; PBS, phosphate-buffered saline; ReGlu, pteroylglutamic acid, folic acid; H,PteGlu,, tetra- DATHF.
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