Abstract
Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic ( approximately 67% of total) and mitochondrial ( approximately 22%) fractions. A polyclonal antipeptide antibody (430Ab) to human FPGS specifically recognized distinct immunoreactive bands ( approximately 60 kDa) present in each subcellular fraction. Human cytosolic FPGS (hcFPGS) migrated more rapidly than mitochondrial FPGS (hmFPGS); their estimated difference in molecular mass was 1 kDa. The human K562 acute nonlymphocytic leukemia and the A253 and FaDu head and neck cancer cell lines also expressed the two FPGS isoforms, and the ratio of hcFPGS to hmFPGS protein in each cell line was similar. Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis (despite having similar levels of total FPGS activity expression), these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity. Higher expression of hmFPGS relative to hcFPGS was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity.
Highlights
Folylpolyglutamate synthetase (FPGS) activity in CCRF-CEM human leukemia cells was found in the cytosolic (Ϸ67% of total) and mitochondrial (Ϸ22%) fractions
Since K562 and A253 cells are intrinsically resistant to pulse methotrexate (MTX) exposure relative to CCRF-CEM and FaDu cells, respectively, because of decreased MTX polyglutamate synthesis, these data suggest that the natural difference in drug sensitivity cannot be explained by compartmentalization of FPGS activity
Higher expression of hmFPGS relative to Human cytosolic FPGS (hcFPGS) was observed in some sublines of CCRF-CEM with acquired MTX resistance suggesting that differential expression of the hmFPGS isoform may contribute to MTX resistance caused by decreased FPGS activity
Summary
Materials—Bovine heart cytochrome c (type Va), NADH, phenylmethylsulfonylfluoride, IGEPAL CA-630 ((octylphenoxy)polyethoxyethanol), and Lubrol PX were from Sigma. The supernatant (cytosolic fraction) was removed for assay of FPGS, lactate dehydrogenase (LDH), and cytochrome c oxidase (cyt c oxidase) and for Western immunoblot analysis. Extraction buffer (100 mM Tris-HCl, pH22 °C 8.85, 0.1 mM Na2EDTA, pH22 °C 8.85, 1 mM benzamidine-HCl, 0.5 mM Pefabloc, 50 mM 2-mercaptoethanol; 0.6 ml/108 cell equivalents) was added to three pellets, and FPGS activity was released into the supernatant by freezing and thawing twice in a dry ice/methanol bath followed by centrifugation at 35,000 ϫ g for 30 min. Protein extraction buffer (50 mM Tris-HCl, pH 7.6, 120 mM NaCl, 0.5% IGEPAL CA-630, 1 mM benzamidine-HCl, 0.5 mM Pefabloc, 0.75 ml/108 cell equivalents [27]) was added to one pellet to extract samples for Western analysis [19]. FPGS activity in PNS, cytosol, and mitochondrial fractions was linear with respect to time and enzyme concentration under the conditions tested. Since this base-eluted antibody was obtained in smaller amounts, it was used only in selected experiments despite its greater specificity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
