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ABSTRACT |
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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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LETTER |
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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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REFERENCES |
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1.
Boos, J.,
G. Werber,
E. Ahlke,
P. Schulze-Westhoff,
U. Nowak-Göttl,
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Monitoring of asparaginase activity and asparagine levels in children on different asparaginase preparations.
Eur. J. Cancer
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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
3.
Kiriyama, Y.,
M. Kubota,
T. Takimoto,
T. Kitoh,
A. Tanizawa,
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and
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Biochemical characterization of U937 cells resistant to L-asparaginase: the role of asparagine synthetase.
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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 |
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.
REPLY
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Abstract
Letter
References
Sheldon M. Schuster Michael S. Kilberg University of Florida Gainesville, FL 32610 |
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