Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

Abstract

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, tsO25C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, tsO25C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20. Amino acid accumulation in mammalian cells is carried out by several distinct transport systems. The major components for neutral amino acid uptake in mammalian cells are systems A, ASC, and L, which were first identified in Ehrlich ascites tumor cells (5, 26). System A is Na+ dependent and serves mainly for the uptake of amino acids with short, polar, or linear side chains. System L is Na+ independent, shows reactivity toward branched-chain and aromatic amino acids, and exhibits rapid exchange between intracellular and extracellular substrates. System ASC is Na+ dependent and shows a strong preference for alanine, serine, and cysteine. Similar systems have been characterized in a number of different cell lines (1,4,9,13,16,27). Recently, transport systems A, ASC, and L have been characterized in Chinese hamster ovary (CHO) cells (2,34). Studies on system L regulation has also been carried out in these cells (24,33,(35)(36)(37) by using a temperature-sensitive leucyl-tRNA synthetase mutant, the CHO tsHl line. When this mutant is incubated at marginally permissive temperatures, a significant increase in leucine transport activity occurs, indicating that the cell responds to leucine limitation resulting from the defective leucyl-tRNA synthetase. Elevated leucine levels in the growth medium, however, allow CHO tsHl cells to grow at nonpermissive temperatures, indicating that increased uptake of leucine is associated with temperature resistance in this mutant. In ad...

Neutral amino acid transport systems in animal cells: Potential targets of oncogene action and regulators of cellular growth

“…Suppression of the temperature-sensitive phenotype with cell fusions to human leukocytes has localized the tRNN eu synthase gene to chromosome 5 and the gene encoding the System L carrier protein to chromosome 20 (Lobaton, Moreno & Oxender, 1984). These studies are in agreement with the postulate of Moore et al (1977) who suggested that the regulation of system L is dependent on the concentration of charged tRNA le" molecules.…”

A genetic approach to the study of neutral amino acid transport in mammalian cells in culture

“…Using a hybrid between CHO ts025C1 containing a temperature-sensitive leucyl t-RNA synthetase and normal human leukocytes, it has been possible to identify a gene on human chromosome 20 that codes for a component that produces increased L-system activity in the hybrid [30]. The hybrid has the maximum derepressed level of the L system as exhibited at moderate nonpermissive temperatures by the ts parent and is not further derepressible.…”

“…Transport of alanine in the absence of Na + is subtracted from that obtained with alanine in the presence of saturation amount of Me?lIB [30,48,51]. It is recognized by these investigators that this assay procedure might include other MeAIB resistant, Na+-dependent transport systems.…”

“…Although proline may be metabolized by CHO cells, under conditions of our assay, there was negligible loss of proline due to metabolism. Other investigators have used a similar procedure, as described above, but have employed AIB instead of proline [30,48,51]. Uptake of AIB on occasion has been employed directly to measure the A system [11].…”

Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli.

“…Expression of leucine transport proteins in E. coli may also be involved in regulating leucyl-tRNA synthetase because a mutant with an apparent deficiency in leucine transport displays a 3-fold elevation in enzyme level (8). Several lines of evidence also suggest a role for leucyl-tRNA synthetase in regulating leucine transport in Chinese hamster ovary cells (9)(10)(11)(12)(13). We report here that sequences in the potential nucleotide-binding domain of leucyl-tRNA synthetase display a high degree of sequence and structural similarity to the carboxyl-terminal domain of the leucine-specific binding protein.…”