Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Abstract

l-Asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal. l-Asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal. Asparaginase is used in the treatment of both pediatric and adult forms of acute lymphoblastic leukemia (ALL) 2The abbreviations used are: ALL, acute lymphoblastic leukemia; mTOR, mammalian target of rapamycin; S6K1, 70-kDa ribosomal protein S6 kinase; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; eIF2, eukaryotic initiation factor 2; GCN2, general control non-derepressible 2; ER, endoplasmic reticulum; PEK/PERK, pancreatic eIF2 kinase/PKR-like endoplasmic reticulum resident kinase, PKR, double-stranded RNA-dependent protein kinase; CHOP/GADD153, CAAT/enhancer-binding protein homologous protein/growth arrest DNA damage-inducible gene 153; BW, body weight; ASNase, asparaginase. 2The abbreviations used are: ALL, acute lymphoblastic leukemia; mTOR, mammalian target of rapamycin; S6K1, 70-kDa ribosomal protein S6 kinase; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; eIF2, eukaryotic initiation factor 2; GCN2, general control non-derepressible 2; ER, endoplasmic reticulum; PEK/PERK, pancreatic eIF2 kinase/PKR-like endoplasmic reticulum resident kinase, PKR, double-stranded RNA-dependent protein kinase; CHOP/GADD153, CAAT/enhancer-binding protein homologous protein/growth arrest DNA damage-inducible gene 153; BW, body weight; ASNase, asparaginase. 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Cancer Res. 1980; 40: 1125-1129PubMed Google Scholar). These studies suggest that depletion of glutamine may be one reason for asparaginase toxicity. Amino acid deprivation is known to inhibit growth and protein synthesis via increased phosphorylation of the α subunit of the translation factor, eukaryotic initiation factor 2 (eIF2). In response to nutrient depletion, phosphorylation of eIF2 by the GCN2 kinase reduces global translation, allowing cells to conserve resources and initiate a reconfiguration of gene expression to either alleviate cellular conditions of stress or trigger programmed cell death (19Kimball S. Anthony T. Cavener D. Jefferson L. Winderickx P. Taylor J Topics in Current Genetics: Nutrient-induced Responses in Eukaryotic Cells. Springer-Verlag, Berlin2004: 113-130Google Scholar, 20Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar). Transcription factors whose expression increases in response to amino acid starvation includes asparagine synthetase and the CAAT/enhancer-binding protein homologous protein (CHOP, also known as growth arrest DNA damage-inducible gene 153, GADD153) (21Gong S. Guerrini L. Basilico C. Mol. Cell. Biol. 1991; 11: 6059-6066Crossref PubMed Scopus (81) Google Scholar, 22Bruhat A. Averous J. Carraro V. Zhong C. Reimold A. Kilberg M. Fafournoux P. J. Biol. Chem. 2002; 227: 48107-48114Abstract Full Text Full Text PDF Scopus (65) Google Scholar, 23Hutson R.G. Kitoh T. Moraga Amador D.A. Cosic S. Schuster S.M. Kilberg M.S. Am. J. Physiol. 1997; 272: C1691-C1699Crossref PubMed Google Scholar, 24Abcouwer S.F. Schwarz C. Meguid R.A. J. Biol. Chem. 1999; 274: 28645-28651Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Microarray analysis of mRNA from leukemic cells treated with asparaginase report induction of both asparagine synthetase and CHOP. Expression of asparagine synthetase was considered experimentally for many years both a marker of asparaginase efficacy and developing drug resistance, but recent work has challenged the concept of using asparagine synthetase as a diagnostic indicator (25Appel I.M. den Boer M.L. Meijerink J.P. Veerman A.J. Reniers N.C. Pieters R. Blood. 2006; 107: 4244-4249Crossref PubMed Scopus (59) Google Scholar, 26Krejci O. Starkova J. Otova B. Madzo J. Kalinova M. Hrusak O. Trka J. Leukemia. 2004; 18: 434-441Crossref PubMed Scopus (53) Google Scholar, 27Stams W.A. den Boer M.L. Beverloo H.B. Meijerink J.P. Stigter R.L. van Wering E.R. Janka-Schaub G.E. Slater R. Pieters R. Blood. 2003; 101: 2743-2747Crossref PubMed Scopus (136) Google Scholar). There are no data examining asparagine synthetase or CHOP levels in non-tumor tissues of whole organisms treated with asparaginase. Dietary amino acid deprivation also mediates translational control via the mammalian target of rapamycin (mTOR) kinase. These events include decreased phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP1) and ribosomal protein S6 kinase (S6K1) that function to stimulate mRNA translation and regulate cell size (28Shah O. Anthony J. Kimball S. Jefferson L. Am. J. Physiol. 2000; 279: E715-E729Crossref PubMed Google Scholar) (917). Asparaginase has been suggested to inhibit S6K1 phosphorylation in leukemic cell lines (29Iiboshi Y. Papst P.J. Hunger S.P. Terada N. Biochem. Biophys. Res. Commun. 1999; 260: 534-539Crossref PubMed Scopus (39) Google Scholar). Yet, although the effects of rapamycin in the whole animal are well documented (30Mohi M. Boulton C. Gu T.-L. Sternberg D. Neuberg D. Griffin J. Gilliland D. Neel B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3130-3135Crossref PubMed Scopus (232) Google Scholar, 31Reiter A. Anthony T. Anthony J. Jefferson L. Kimball S. Int. J. Biochem. Cell Biol. 2004; 36: 2169-2179Crossref PubMed Scopus (64) Google Scholar, 32Anthony J. Yoshizawa F. Anthony T. Vary T. Jefferson L. Kimball S. J. Nutr. 2000; 130: 2413-2419Crossref PubMed Scopus (616) Google Scholar), there are no studies reporting the effects of asparaginase on mTOR signaling in vivo. Although the inhibition of protein synthesis has been implicated as the basis for altered function of several non-target tissues following asparaginase treatment, mechanisms by which asparaginase reduces protein synthesis remain unknown. Herein we report comparative effects of two forms of asparaginase (differing in the ability to degrade glutamine) on the regulation of protein synthesis in tissues that are associated with secondary complications, namely the liver, pancreas, and spleen. This study is the first to show tissue-specific changes in protein synthesis, eIF2α phosphorylation, and mTOR signaling following asparaginase treatment in the whole animal. We also show that prevention of glutamine depletion by the use of a novel glutaminase-free enzyme prevents some but not all measured stress responses elicited by a contemporary asparaginase. Specifically, both asparagine synthetase and CHOP are induced by both enzymes, in an apparent eIF2 phosphorylation-independent manner. Finally, unlike dietary amino acid deprivation, phosphorylation of eIF2 following asparaginase is initially but not solely dependent on GCN2. These data provide important insight into the cellular stress mechanisms elicited by asparaginase and support testing of Wolinella asparaginase in chemotherapeutic regimens to treat ALL. Measurement of l-Asparaginase Activity—The activity of experimental l-asparaginase derived from Escherichia coli (Elspar® product from Merck, passed thru a gel-filtration column to remove residual endotoxin as described (33Loos M. Vadlamudi S. Meltzer M. Shifrin S. Borsos T. Goldin A. Cancer Res. 1972; 32: 2292-2296PubMed Google Scholar)) or W. succinogenes (prepared and purified under GMP standards within the NIH/NCI-RAID Developmental Therapeutics program) was determined by the Nesslerization technique, as previously described (17Distasio J.A. Niederman R.A. Kafkewitz D. Goodman D. J. Biol. Chem. 1976; 251: 6929-6933Abstract Full Text PDF PubMed Google Scholar, 34Broome J.D. J. Natl. Cancer Inst. 1965; 35: 967-974PubMed Google Scholar). Briefly, the production of ammonia by l-asparaginase over time was expressed relative to the slope of known ammonia standards. The resulting value represented the activity of the enzyme in international units (IUs), in which one IU equaled the amount of enzyme that catalyzed the formation of 1 μmol of ammonia per min. Enzyme activity of l-asparaginase was determined prior (<1 h) to administration. Animals—The following study protocol was approved by the Institutional Care and Use Committee at the Indiana University School of Medicine-Evansville. Six to 8 week old male and female C57BL/6J mice (also referred to as GCN2+/+) and GCN2 null mice (also referred to as GCN2–/–; backcrossed onto the C57BL/6J genetic background 8–10 generations (35Anthony T.G. McDaniel B.J. Byerley R.L. McGrath B.C. Cavener D.R. McNurlan M.A. Wek R.C. J. Biol. Chem. 2004; 279: 36553-36561Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar)) were maintained on a 12-h light:dark cycle and provided free access to commercial rodent chow (PMI International, Brentwood, MO) and tap water prior to the experiment. On the day of the experiment, mice were given a single injection of phosphate-buffered saline that contained an enzyme activity of 0, 1.5, or 3.0 IU of l-asparaginase (from E. coli or Wolinella) per gram body weight. It is reported that mice are resistant to l-asparaginase immunosuppression and hepatotoxicity up to 2.0 IU/g (36Reiff A. Zastrow M. Sun B.C. Takei S. Mitsuhada H. Bernstein B. Durden D.L. Clin. Exp. Rheumatol. 2001; 19: 639-646PubMed Google Scholar), so these doses were chosen to represent enzyme activities below and above this threshold. All mice were injected in the morning and allowed free access to food and water throughout the day. At the indicated times (usually 6 h later but 15 min and 1 h were also studied), mice were euthanized by decapitation, and the liver, pancreas, and spleen were dissected carefully, rinsed in ice-cold phosphate-buffered saline, weighed, and frozen immediately in liquid nitrogen. Trunk blood was collected to obtain serum for analysis of amino acid profiles. In a separate experiment, C57BL/6J mice were given a single intraperitoneal injection of 200 mg/kg ammonium chloride or 0 or 3 IU/g BW E. coli asparaginase and euthanized 30 min later. Blood was collected to measure serum ammonia concentrations, and the liver was examined for phosphorylation of eIF2α (see below for description of analyses). Amino Acid Profiles—Serum was obtained by centrifugation of clotted blood and then snap-frozen and stored at –20 °C. Serum samples were sent to the Indiana University School of Medicine Quantitative Amino Acid Core Facility (under the direction of Dr. Edward Liechty) for the determination of amino acid profiles by the ninhydrin method, using standard ion-exchange chromatography with a Beckman 6300 automated amino acid analyzer. Tissue Amino Acid Concentrations—Frozen powdered tissue was incubated in 3% perchloric acid on ice and then centrifuged for 10 min at 10,000 × g. The collected supernatant was applied onto a cation exchange column (Dowex AG 50W-X8 resin, 100–200 mesh hydrogen form, Bio-Rad), eluted with ammonium hydroxide, and dried to completion using a Savant evaporator. Free amino acid analysis was performed using a Waters 2690 high-performance liquid chromatographic separation module (Waters, Milford, MA) and 474 fluorescence detector with pre-column derivatization (ortho-phthalaldehyde/3-mercaptopropionic acid). The internal standard used was methionine sulfone. The separation column used was a Synergi 4μ MAX-pro, 250 × 4.6 mm (Phenomenex, Torrance, CA) heated at 40 °C. Plasma Ammonia—Ammonia concentrations were measured using a commercial enzymatic method (Diagnostic Chemicals Limited, Oxford, CT). Duplicate 100-μl samples were added to 3.0 ml of a buffer mixture that contained (in mm) 2.2 ADP, 3.5 α-ketoglutarate, and 0.2 NADPH. Subsequent addition of 20 μl of glutamate dehydrogenase (1200 IU/ml) catalyzes the formation of glutamate from glutamine and oxidation of NADPH to NADP+ at room temperature. Absorbance at 340 nm was measured before the addition of enzyme (t = 0) and following reaction completion (t = 6 min). The change in absorbance was used to calculate plasma ammonia concentration using ammonia standards and blanks assayed alongside samples. Quantitative Reverse Transcription-PCR of Asparagine Synthetase—Total RNA was extracted from frozen tissue using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) followed by DNase treatment (VersaGene DNase kit, Gentra Systems). The A260/280 ratio was between 1.8 and 2.0 following RNA clean-up. mRNA expression was determined by quantitative real-time PCR using TaqMan chemistry. The relative expression levels of asparagine synthetase mRNA was determined using the Eurogentec RTqPCR mastermix (Eurogentec, Belgium) and ABI PRISM 7700 Sequence Detection System. The PCR mix contained 1× master mix and 0.125 μl of Euroscript+RT and RNase Inhibitor (reverse transcription, 0.125 unit/μl and RNase inhibitor, 0.05 unit/μl). Asparagine synthetase and β-actin primers and probes were added at final concentrations of 200 nm and 100 nm, respectively. The primers used for asparagine synthetase were: forward, 5′-GGAGAGGGGTCAGATGAACTT-3′ and reverse, 5′-CTCCTCCTCGGCCTTCTC-3′. 1 μg of total RNA was used per reaction in a 25-μl reaction volume. All samples were run in duplicates. The thermal cycler conditions were 48 °C for 30 min, 95 °C for 10 min, and 45 cycles of 95 °C for min and °C for 1 min. were using Sequence Detection were obtained as is to the was used as gene and expression of asparagine synthetase gene in samples treated with 3 units of was in to control samples using the method were expressed as change with to the experimental The data were by with and was at before were mice were injected with a of and with MA) for the of tissue protein synthesis as previously described T. McNurlan M. C. P. Am. J. Physiol. Google Scholar, M. H. L. L. Physiol. 1991; PubMed Scopus Google Scholar). tissue was to the of into liver protein as previously described G. G. P. M. Am. J. Physiol. 2002; Google Scholar). 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Chem. 2004; 279: 36553-36561Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) using a in of buffer of (in acid 100 0.2 2 1 1 and The were immediately centrifuged at 10,000 × for 10 min at °C for analysis of protein expression and phosphorylation as described of of eIF2α was using an that the protein only is at Inc., were for total eIF2α with an that the protein of phosphorylation samples were in a buffer mm mm mm mm and 1 mm with of 1 and 1 and centrifuged for 10 min at 10,000 × g. of protein concentration were added to buffer and onto for by was measured as a in as by using a R. Wek R.C. Biochem. J. 2000; PubMed Scopus Google Scholar). of of was measured as a change in as by analysis as described previously T. A. Anthony J. Kimball S. Jefferson L. Am. J. Physiol. 2001; PubMed Google Scholar). Briefly, an of the 10,000 × supernatant was for 10 min and centrifuged at 10,000 × for 30 min at °C. The supernatant was added to buffer and to protein analysis using a of of S6K1 was measured as a in as described previously T. A. Anthony J. Kimball S. Jefferson L. Am. J. Physiol. 2001; PubMed Google Scholar). Briefly, an of the 10,000 × supernatant was added to analysis was then performed using a S6K1 Expression of × supernatant of protein concentration were added to buffer and onto for by proteins were onto and analysis was then performed using a data were by the for the were using analysis of to with drug and as the a significant was among treatment were with The of was at 0.05 for all Although asparaginase has been an of induction therapy for over 40 of action This is with to the basis of using asparaginase are not to efficacy with this J. J. Pediatr. Hematol. Oncol. 2004; 26: 273-274Crossref PubMed Scopus (9) Google Scholar). are by data on the and of the because to the of action by of not been J.P. J. J. 2004; PubMed Scopus Google Scholar). of the of asparaginase and Glutamine in and following a of l-Asparaginase from E. coli but W. both forms of asparaginase reduced the concentration of asparagine in the blood below the levels of at both enzyme doses a the amount of acid increased in the of all mice of or form of enzyme Asparaginase from E. coli glutaminase activity, which is reported to the of glutamine at the of asparagine E. Cancer Res. 35: Google Scholar). with this treatment with E. coli asparaginase reduced circulating glutamine concentrations, resulting in and increases in serum acid in the and 3.0 IU/g and In contrast, treatment with Wolinella asparaginase not serum although a significant in circulating glutamate was at the of for this result is that even glutamine is not by in the body is increased in response to asparagine depletion. of the asparagine synthetase glutamine provide the to asparagine, to a production of glutamate in the Although no changes in were enzyme circulating ammonia increased following of the of E. coli asparaginase and In contrast, treatment with Wolinella not ammonia There were no changes in serum concentrations of any of the essential or amino by either enzyme not synthesis in the liver pancreas and spleen 6 h following a single intraperitoneal injection of l-asparaginase at 0, 1.5, or 3.0 BW from E. coli or are expressed as of into