John F. Ash

Inducible expression of the GLT-1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake

Inducible expression of the mammalian glial cell glutamate transporter GLT‐1 has been established in a CHO cell line selected for low endogenous Na+‐dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline‐containing medium, to activate GLT‐1 expression via the Tet‐On system, increased uptake of the GLT‐1 substrate d‐aspartate 280‐fold, and increased cell size. Applying glutamate to whole‐cell clamped, doxycycline‐treated cells evoked a transporter‐mediated current with characteristics appropriate for GLT‐1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT‐1, the most abundant glutamate transporter in the brain.

The Na+-Dependent Glutamate and Aspartate Transporter Supports Glutathione Maintenance and Survival of CHO-K1 Cells

Glutathione synthesis, a vital cellular process, depends on L-cystine uptake by the amino acid transporter, System xC-. Here we show that a second transporter, System XAG-, is required for normal System xC-activity and glutathione maintenance by employing somatic cell mutants of CHO-K1. Uptake by System xC-in two XAG--null mutants is significantly lower than that of CHO-Kl, either under control conditions or after prolonged treatment with an electrophile. In addition, levels of glutathione in control and treated mutant cells are less than half those of wild-type CHO-K1 or of a pseudorevertant. The significance of this reduction was tested by chemical challenge: mutants are twofold more sensitive than wild type to reactive oxygen species generated by phenylbenzoquinone and to damage produced by the anticancer drug, cisplatin. These results suggest that System XAG-provides a significant portion of the glutamate used to energize the uptake of cystine required for the synthesis of glutathione.

New Mutations and Phenotypes Associated with Glutamate and Aspartate Transport in Chinese Hamster Ovary (CHO-K1) Cells

Two new Chinese hamster ovary cell (CHO-K1) mutants lacking amino acid transport System XAG− activity were isolated by [3H]aspartate suicide selection. These null mutants, Dd-B6 and Dd-B7, were analyzed by somatic cell hybridization, along with previously described partial-function mutants, Ed-A1 and Ed-B8. With respect to System XAG− activity, all four mutations fell into a single complementation group. By quantitative assay, the mutations in Ed-A1 and Ed-B8 behaved as simple recessives in fusions with wild type cells, while those in Dd-B6 and Dd-B7 were codominant. We have discovered that Ed-A1 and Ed-B8 are highly permeable to smal neutral molecules. This high permeability phenotype was dominant to wild-type. Northern, Southern, and Western analyses indicated that System XAG− in CHO is not closely related to any of the three well characterized glutamate transporters represented by GLT-1, EAAC1 or GLAST.

Novel regulations of glutamate and aspartate uptake by HeLa cells

Pathways of L-glutamate and L-aspartate import by HeLa S3 cells were investigated before and after the cells were depleted of internal amino acids by starvation. Two new regulations of transport were observed in starved cells. Aspartate entered nonstarved cells by two routes, one non-saturable and one, an apparent analog of saturable system X-AG, that was sodium-dependent and competitively inhibited by glutamate. Starvation for one hour in saline increased the efficiency of saturable aspartate import, increasing Vmax and decreasing Km, an effect not previously reported for system X-AG. Glutamate uptake by nonstarved cells appeared to occur through system X-AG; through an analog of system X-C, which was sodium-independent, cystine- and quisqualate-inhibitable; as well as through one or more nonsaturable pathways. Starvation in saline for one hour resulted in the appearance of a new low-affinity saturable glutamate uptake system. This new system was sodium-dependent but not inhibited by aspartate.

Selection of Chinese hamster ovary cells (CHO-K1) with reduced glutamate and aspartate uptake

Transport of L-[3H]glutamate into Chinese hamster ovary cells (CHO-K1) was characterized and the results used to design a tritium suicide selection for cells with transport defects. Replicas of surviving colonies on polyester cloth disks were scrrened by autofluorography for reduced uptake and two mutant clones, Ed-A1 and Ed-B8, were obtained. Uptake of glutamate through a sodium-dependent system in both mutants was characterized by significant reductions inVmax and increases inKm compared to parental cells, but their response to removal of extracellular sodium differed, suggesting distinct mutations in the two lines. TheVmax of aspartate uptake through this system was reduced in both mutants, to one-ninth in the case of Ed-B8. Glutamate uptake through a sodium-independent system was not altered in either mutant. Surprisingly, acid-soluble intracellular pools of several amino acids were higher in both mutants.

Numerical analysis reveals complexities of glutamate transport

The uptake of radiolabeled glutamate into cultured human (HeLa S3) and hamster (CHO-K1) cells was analyzed according to modified Michaelis-Menten models fit by the Marquardt least-squares method. Kinetic parameters not previously reported for these cells were obtained. Some rarely used features available with this fitting method proved to be extremely helpful. Most importantly, a goodness-of-fit measure revealed a significant alteration of glutamate uptake in HeLa cells that was induced by starvation. This apparent regulation, unexpected for glutamate transport, might have been missed if the fit had been judged by eye or by the magnitude of parameter standard deviations. Techniques for analyzing parameter distributions, improving experimental design and performing tests of significance are also described.

Selection of a lysine-resistant CHO-K1 mutant with reduced amino acid transport through multiple systems

High levels ofl-lysine were used to select for resistant variants of Chinese hamster ovary (CHO-K1) cells. Surviving colonies were screened for altered lysine transport and two with reduced uptake were picked. Clone CH-Kr, derived from the more severely affected colony, was analyzed in detail. In starved cells theVmax of lysine uptake in CH-Kr was half that of CHO whileKm was unaltered. The intracellular pool of lysine, a substrate of cationic amino acid transport system y+, was significantly lower in CH-Kr. However, transport and pools of other amino acids, which are not substrates of y+, were also reduced in CH-Kr, as was the internal sodium concentration, while hexose import was increased. It appears that the mutation in CH-Kr is pleiotropic, affecting some general aspects of amino acid transport.

A Rapid in Vitro Assay for Detecting Phorbol Ester-Like Tumor Promoters

We are developing a rapid in vitro assay for detecting compounds with phorbol ester-like tumor promotion activity. This assay is based on the potentiation of the cytotoxic effect of ouabain by phorbol esters such as Tetra-O-decanoyl, 13-phorbol acetate (TPA) and 3-O-tetradecanoy ingenol (3TI). Ouabain is a specific inhibitor of the Na+, K+ -ATPase, a plasma membrane enzyme responsible for the active maintenance of the sodium and potassium gradients across the cell membrane.