Letters to the Editor

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The following is the abstract of the article discussed in the subsequent letter:

Hutson, Richard G., Toshiyuki Kitoh, David A. Moraga Amador, Sanja Cosic, Sheldon M. Schuster, and Michael S. Kilberg. Amino acid control of asparagine synthetase: relation to asparaginase resistance in human leukemia cells. Am. J. Physiol. 272 (Cell Physiol. 41): C1691-C1699, 1997.---Complete amino acid deprivation in mammalian cells causes a significant enhancement in gene expression for a number of important cellular activities; among these is asparagine synthetase (AS). The data presented demonstrate that, in both nonleukemic (rat Fao hepatoma cells) and human leukemia cells (MOLT-4, NALL-1, and BALL-1), AS mRNA levels, protein content, and enzymatic activity are induced after incubation in an otherwise complete tissue culture medium that is deficient in a single amino acid or in medium that has been depleted of the amino acid asparagine by the addition of asparaginase. Complete amino acid deprivation results in a concerted increase in AS mRNA, protein, and enzymatic activity, which, in conjunction with previously published research, suggests that the mechanism of this cellular response involves transcriptional control of the AS gene. Asparaginase treatment is a standard component of acute lymphoblastic leukemia therapy for which the effectiveness is related to the inability of these cells to upregulate AS activity to a sufficient level. With regard to the asparaginase sensitivity of the three human leukemia cell lines, there was a trend toward an inverse relation to the degree of AS expression. Selection for asparaginase-resistant MOLT-4 sublines resulted in enhanced AS mRNA and protein content regardless of whether the cells had been selected by asparaginase treatment directly or asparagine was removed from the culture medium. Collectively, the data illustrate that further advances in asparaginase therapy will require additional knowledge of amino acid-dependent regulation of AS gene expression and, conversely, that asparaginase resistance represents a model system for investigating metabolite control in a clinically relevant setting.

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Unphysiological effects contributing to asparaginase toxicity in vitro

To the Editor: Asparaginase has long been accepted as a major cytotoxic agent in the treatment of acute lymphatic leukemias and has been the subject of controversy due to its large range of side effects, but knowledge about asparagine synthetase (AS), the apparent source of effects as well as side effects, is unsatisfactory. The extensive study by Hutson et al. (2), therefore, engages strong interest from clinicians. The results demonstrate that the reduction of Asn or addition of asparaginase increased the cellular AS activity, the AS mRNA, and the protein content. The activity of AS was inversely related to the sensitivity to asparaginase in human leukemia cells. There remained, however, the observation that AS expression increased sevenfold, whereas the resistance to asparaginase increased >1,000 times. The authors propose that this may be influenced by additional factors that contribute to asparaginase resistance. Interestingly, the resistance toward the enzyme asparaginase differed between MOLT4/Rasn (Asn-free growing cells) and MOLT4/Rasp (cells cultured with asparaginase). In line with these results, findings in a highly asparaginase-sensitive human histiocytic lymphoma cell line, U-937, showed an either 80- or 7-fold increase of AS activity in cells whose resistance was produced by asparaginase or by Asn deprivation, respectively (3).

According to Hutson et al. (2), even differences in the content of His, an amino acid independent from asparaginase, resulted in an increase in cellular AS. Because asparaginase has significant inherent glutaminase activity, Gln serves as a substrate as well as Asn, especially in Asn-depleted medium. The main products are Asp, Glu, and ammonia. Additional cytotoxic effects of asparaginase may thus be due to alterations in the culture medium. In our experiments, RPMI 1640 medium was incubated with various concentrations of asparaginase (0-10,000 U/l). Asparaginase activity and Asn, Asp, Gln, Glu, and ammonia were monitored for 24 h (4). In asparaginase-free controls, all parameters remained constant. Even at a 2 U/l concentration of asparaginase, however, the medium was completely Asn free within 4 h. The asparaginase concentration, therefore, does not influence even the exposure to Asn in 96-h assays. At higher concentrations (200-2,000 U/l), Glu and ammonia preferentially increased in an exponential manner, whereas Asn remained deficient and Asp was constant. There was a clear-cut relationship between asparaginase activity and the 24-h area under the data of Gln (inverse), Glu, and ammonia but not for Asn itself. The conditions, however, are not comparable with the in vivo situation (1), where the various products and substrates are all part of metabolic pathways and equilibrium conditions.

These observations may help to explain the gap between the slight increase in AS expression compared with the much more impressive increase in asparaginase resistance in resistant cell lines observed by Hutson et al. (2). In summary, in vitro cytotoxicity assays involving asparaginase should be interpreted with caution regarding their significance in vivo, especially if they are intended to contribute to therapy decisions.

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1.   Boos, J., G. Werber, E. Ahlke, P. Schulze-Westhoff, U. Nowak-Göttl, E. Verspohl, J. Ritter, and H. Jürgens. Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations. Eur. J. Cancer 32A: 1544-1550, 1996.

2.   Hutson, R. G., T. Kitoh, D. A. Moraga Amador, S. Cosic, S. M. Schuster, and M. S. Kilberg. Amino acid control of asparagine synthetase: relation to asparaginase resistance in human leukemia cells. Am. J. Physiol. 272 (Cell Physiol. 41): C1691-C1699, 1997[Abstract/Free Full Text].

3.   Kiriyama, Y., M. Kubota, T. Takimoto, T. Kitoh, A. Tanizawa, Y. Akiyama, and H. Mikawa. Biochemical characterization of U937 cells resistant to L-asparaginase: the role of asparagine synthetase. Leukemia 3: 294-291, 1989[Medline].

4.   Wagner, A., P. Schulze-Westhoff, H. Jürgens, and J. Boos. In vitro monitoring of asparaginase: unphysiological alteration of culture medium. In: Experimental Approaches and Novel Therapies. Acute Leukemias, edited by W. Hiddemann, T. Büchner, B. Wörmann, J. Ritter, U. Creutzig, M. Keating, and W. Plunkett. Berlin, Germany: Springer, 1998, vol. VII, p. 570-574.

Alexandra Wagner
Joachim Boos
Department of Pediatric Oncology
University of Münster
D-48149 Münster, Germany

    REPLY
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To the Editor: We agree that the cytotoxicity assays reported in our paper may be able to be performed at times <94 h, but we do not believe that the time period studied negates the validity of the results, as shown bythe BALL-1 cells. These cells were known to have a relative resistance to L-asparaginase, and our cytotoxicity data confirm those observations. We also agree that the glutaminase activity associated with the enzyme used therapeutically may contribute to the asparaginase toxicity and the development of asparaginase resistance, both in vitro and in vivo. Clearly, additional investigation is needed to understand fully the mechanisms responsible for asparaginase resistance.

Sheldon M. Schuster
Michael S. Kilberg
University of Florida
Gainesville, FL 32610 


AJP Cell Physiol 274(4):C1185-C1185
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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