Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

Andrulis, Irene L.; Duff, C; Evans-Blackler, S; Worton, R; Siminovitch, Louis

doi:10.1128/mcb.3.3.391

Cited by 34 publications

(10 citation statements)

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“…We have used this drug previously to isolate mutants of human and CHO cells (2) and have found that VOL. 7, 1987 on May 11, 2018 by guest http://mcb.asm.org/ Downloaded from albizziin resistance behaves as a dominant selectable marker in animal cells.…”

Section: Discussionmentioning

confidence: 99%

“…The regulation of AS has been studied in cultured mammalian cells (for a review, see reference 1), and overproducing cell lines, some with amplified copies of the gene, have been isolated in response to drug selection (2,3,6,11). Most mammalian cells express AS and regulate the level of activity in response to the concentration of asparagine in the medium and the extent of tRNA aminoacylation (4,7).…”

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confidence: 99%

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Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Andrulis¹,

Chen²,

Ray³

1987

Mol. Cell. Biol.

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Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.Asparagine synthetase (AS) is a housekeeping enzyme responsible for the biosynthesis of asparagine from glutamine and aspartic acid. The regulation of AS has been studied in cultured mammalian cells (for a review, see reference 1), and overproducing cell lines, some with amplified copies of the gene, have been isolated in response to drug selection (2, 3, 6, 11). Most mammalian cells express AS and regulate the level of activity in response to the concentration of asparagine in the medium and the extent of tRNA aminoacylation (4, 7). In contrast to normal cells, some tumor cells exhibit little or no AS activity and rely on the surrounding medium as a source of exogenous asparagine. This property has been exploited in the treatment of certain cancers, since the tumor cells can be selectively killed by the chemotherapeutic drug asparaginase which hydrolyzes circulating asparagine (9, 24). One of the most sensitive human malignancies was found to be childhood acute lymphoblastic leukemia, and asparaginase is now routinely used in induction and consolidation protocols for patients with this disease (23). In mouse model systems, it was found that asparaginase-sensitive tumor cells could be made asparaginase-resistant by repeated subculturing of these cells in sublethal concentrations of the drug. The resistant cells were found to have regained AS activity and in some cases had elevated levels of enzyme activity up to 60-fold higher than that in normal cells (13,24). The mechanism responsible for the lack of activity of AS in the sensitive leukemic cells and for the overproduction in the asparaginase-resistant variants is of clinical importance for the treatment of acute lymphoblastic leukemia patients both initially and during relapse.To investigate the molecular basis for the sensitivity to asparaginase in human acute...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Andrulis¹,

Chen²,

Ray³

1987

Mol. Cell. Biol.

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“…It was expected that a low level of amplification would be sufficient to cause MTX resistance, since the dose of MTX that can be safely administered is limited by toxic effects on normal tissues. In the three patients reported (two leukemia [2,3] and one small-cell lung car- Yes [17] Yes [18] Yes [19] (?) [20] (?)…”

Section: E Gene Amplification In Patients With Leukemia Resistant Tomentioning

confidence: 99%

Mechanisms of Drug Resistance in Human Leukemia

Bertino¹,

Srimatkandada²,

Carman³

et al. 1985

Modern Trends in Human Leukemia VI New Results in Clinical and Biological Research Including Pediatric Oncology

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“…Single-step mutant lines resistant to the amino acid analog P-aspartyl hydroxamate (P-AHAR) exhibit elevations in AS activity up to 8-fold over that of the parental cells (6,11), and multistep 1-AHAR lines express as high as 20-fold increases in AS activity (6). On the other hand multistep albizziin-resistant (AlbR) lines that overproduce the enzyme by 300-fold have been isolated (2). We took advantage of a highly resistant multistep line to clone an AS cDNA and show that the gene was amplified in this line (25).…”

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confidence: 99%

“…We have also investigated the role of DNA methylation in the expression of AS activity in drug-resistant mutant cell lines that overproduce the enzyme (2,3,6). Single-step mutant lines resistant to the amino acid analog P-aspartyl hydroxamate (P-AHAR) exhibit elevations in AS activity up to 8-fold over that of the parental cells (6,11), and multistep 1-AHAR lines express as high as 20-fold increases in AS activity (6).…”

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confidence: 99%

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

Andrulis¹,

Barrett²

1989

Mol. Cell. Biol.

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In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild.type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziinresistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in j-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

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Chromosomal alterations associated with overproduction of asparagine synthetase in albizziin-resistant Chinese hamster ovary cells.

Cited by 34 publications

References 12 publications

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Mechanisms of Drug Resistance in Human Leukemia

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

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