Exogenous Asparagine Research Articles

Exploring the potential of asparagine restriction in solid cancer treatment: recent discoveries, therapeutic implications, and challenges.

Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges.

Abstract B012: Differential stress responses drive asparagine exchange in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the United States, contrasting a relatively low incidence rate. A principal barrier to the treatment of pancreatic cancer is the densely fibrotic tumor microenvironment and the high interstitial pressure which acts to collapse blood vessels, impairing the delivery of chemotherapy. This lack of functional vasculature limits nutrient availability within the tumor, causing cancer cells to develop numerous metabolic adaptations to allow for proliferation in hypoxic, austere conditions. Recent studies have demonstrated several clonal behaviors of cancer cells within individual pancreatic tumors, although it is unclear if this is true of metabolism. Exploring this, we found the metabolic profiles and sensitivities of a series of clonal cell lines derived from a single pancreatic tumor clustered into two distinct groups. These groups demonstrated differential sensitivities to inhibitors of mitochondrial metabolism; whereby the growth of the sensitive clones during impaired respiration can be rescued through co-culture with the insensitive cells. Profiling metabolites exchanged by the clonal populations, we identified this is mediated by asparagine released by insensitive cells. Mechanistically, the production of asparagine is driven by differential stress response pathway activation between clones. We further determined that exogenous asparagine supports the growth of both cancer and non-cancerous cells during limited respiration. Conversely, we found that the use of asparaginase to degrade asparagine sensitizes PDA tumors to mitochondrial inhibition. Collectively these data demonstrate distinct metabolic classes of cancer cells within single tumors and identify asparagine as a potential target to sensitize tumors to mitochondrial inhibition. Citation Format: Cecily Anaraki, Galloway Thurston, Nina G. Steele, Samuel A. Kerk, Hanna Hong, Peter Sajjakulnukit, Anthony Andren, Catherine Felton, Alica Beutel, Rima Singh, Heather R. Christofk, Andrea Viale, Howard Crawford, Marina Pasca di Magliano, Claus Jorgensen, Costas A. Lyssiotis, Christopher J. Halbrook. Differential stress responses drive asparagine exchange in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B012.

Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG–asparaginase sensitizes tumors to mitochondrial targeting with phenformin.

Novel Insights on the Use of L-Asparaginase as an Efficient and Safe Anti-Cancer Therapy.

Simple SummaryL-asparaginase (L-ASNase) therapy is key for achieving the very high cure rate of pediatric acute lymphoblastic leukemia (ALL), yet its use is mostly confined to this indication. One main reason preventing the expansion of today’s FDA-approved L-ASNases to solid cancers is their high toxicity and side effects, which become especially challenging in adult patients. The design of optimized L-ASNase molecules provides opportunities to overcome these unwanted toxicities. An additional challenge to broader application of L-ASNases is how cells can counter the pharmacological effect of this drug and the identification of L-ASNases resistance mechanisms. In this review, we discuss recent insights into L-ASNase adverse effects, resistance mechanisms, and how novel L-ASNase variants and drug combinations can expand its clinical applicability, with a focus on both hematological and solid tumors.L-Asparaginase (L-ASNase) is an enzyme that hydrolyses the amino acid asparagine into aspartic acid and ammonia. Systemic administration of bacterial L-ASNase is successfully used to lower the bioavailability of this non-essential amino acid and to eradicate rapidly proliferating cancer cells with a high demand for exogenous asparagine. Currently, it is a cornerstone drug in the treatment of the most common pediatric cancer, acute lymphoblastic leukemia (ALL). Since these lymphoblasts lack the expression of asparagine synthetase (ASNS), these cells depend on the uptake of extracellular asparagine for survival. Interestingly, recent reports have illustrated that L-ASNase may also have clinical potential for the treatment of other aggressive subtypes of hematological or solid cancers. However, immunogenic and other severe adverse side effects limit optimal clinical use and often lead to treatment discontinuation. The design of optimized and novel L-ASNase formulations provides opportunities to overcome these limitations. In addition, identification of multiple L-ASNase resistance mechanisms, including ASNS promoter reactivation and desensitization, has fueled research into promising novel drug combinations to overcome chemoresistance. In this review, we discuss recent insights into L-ASNase adverse effects, resistance both in hematological and solid tumors, and how novel L-ASNase variants and drug combinations can expand its clinical applicability.

Enhanced Antitumor Effect in Liver Cancer by Amino Acid Depletion-Induced Oxidative Stress

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC cells consume large amounts of glutamine to survive, but can adapt to glutamine depletion in the presence of an exogenous asparagine. L-asparaginase (ASNase) converts glutamine and asparagine to glutamate and aspartate, respectively, and has been used to treat leukemia. Here we examined the effects of ASNase treatment on HCC cells and explored the potential impact of combining ASNase with the tyrosine kinase inhibitor lenvatinib (Len) for HCC treatment. Cell viability and death of HCC cell lines treated with either Len or ASNase alone or with Len and ASNase combined were determined. We assessed mRNA and protein expression levels of glutamine synthetase (GS) and asparagine synthetase (ASNS) by real-time quantitative PCR and immunoblotting. The antitumor effect of the combination therapy relative to Len or ASNase monotherapy was also evaluated in a xenograft tumor mouse model. ASNase treatment inhibited growth of SNU387 and SNU398 HCC cells, which have low GS and high ASNS expression levels, respectively, but did not clearly inhibit growth of the other cell lines. Len plus ASNase combination therapy synergistically inhibited proliferation and induced oxidative stress leading to cell death of some HCC cells lines. However, cell death of Huh7 cells, which express ASCT2, an important glutamine transporter for cancer cells, was not affected by the combination treatment. In a xenograft model, Len combined with ASNase significantly attenuated tumor development relative to mice treated with Len or ASNase alone. ASNase-mediated targeting of two amino acids, glutamine and asparagine, which are indispensable for HCC survival, induces oxidative stress and can be a novel cancer treatment option that exerts a synergistic effect when used in combination with Len.

DDRE-04. THE COMBINED TREATMENT OF L-ASPARAGINASE AND 6-DIAZO-5-OXO-L-NORLEUCINE INHIBIT THE PROLIFERATION OF TEMOZOLOMIDE-SENSITIVE OR RESISTANT GLIOBLASTOMA CELLS

Glioblastoma is one of the aggressive brain tumors with a 5-year survival rate of < 10%. The standard treatment is maximal safe resection, followed by radiation therapy and temozolomide (TMZ). Clinically, the resistance to TMZ is a big problem. Cancer cells have been revealed to show different metabolism from normal cells. The object of this study is to evaluate whether cancer metabolism, especially asparagine, could be a new target of treatment in primary and recurrent glioblastoma. Glioblastoma cells (U251 and U87) were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase converts asparagine into aspartate and depletes asparagine. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase. L-asparaginase or DON suppressed the proliferation of U251, and U87 cells in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. The effect was counteracted by exogenous asparagine. The combined treatment induced greater apoptosis and autophagy than did single-drug treatment. Several clones of TMZ-resistant U251 were obtained after long treatment of TMZ to U251. The expression of MSH6, one of the mismatch repair proteins, was suppressed in these resistant clones. The synergistic effect of L-asparaginase and DON was detected in these U251-derived TMZ-resistant clones. These results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with primary and recurrent glioblastoma.

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

L-asparaginase and 6-diazo-5-oxo-L-norleucine synergistically inhibit the growth of glioblastoma cells.

Glioblastoma is an aggressive central nervous system tumor with a 5-year survival rate of < 10%. The standard therapy for glioblastoma is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide. New approaches to treatment of glioblastoma, such as targeting metabolism, have been studied. The object of this study is to evaluate whether asparagine could be a new target for treatment of glioblastoma. We investigated a potential treatment for glioblastoma that targets the amino acid metabolism. U251, U87, and SF767 glioblastoma cells were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase hydrolyzes asparagine into aspartate and depletes asparagine. L-asparaginase has been used for the treatment of acute lymphoblastic leukemia. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase. Cell viability was measured after 72h of treatment. MTS assay showed that L-asparaginase suppressed the proliferation of U251, U87, and SF767 cells in a dose-dependent manner. DON also inhibited the proliferation of these cell lines in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. Exogenous asparagine rescued the effect of inhibition of proliferation by L-asparaginase and DON. The expression of asparagine synthetase mRNA was increased in cells treated with a combination of L-asparaginase and DON. This combined treatment also induced greater apoptosis and autophagy than did single-drug treatment. The results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with glioblastoma.

Asparagine Deprivation Causes a Reversible Inhibition of Human Cytomegalovirus Acute Virus Replication.

As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients.IMPORTANCE HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.

As Extracellular Glutamine Levels Decline, Asparagine Becomes an Essential Amino Acid

When mammalian cells are deprived of glutamine, exogenous asparagine rescues cell survival and growth. Here we report that this rescue results from use of asparagine in protein synthesis. All mammalian cell lines tested lacked cytosolic asparaginase activity and could not utilize asparagine to produce other amino acids or biosynthetic intermediates. Instead, most glutamine-deprived cell lines are capable of sufficient glutamine synthesis to maintain essential amino acid uptake and production of glutamine-dependent biosynthetic precursors, with the exception of asparagine. While experimental introduction of cytosolic asparaginase could enhance the synthesis of glutamine and increase tricarboxylic acid cycle anaplerosis and the synthesis of nucleotide precursors, cytosolic asparaginase suppressed the growth and survival of cells in glutamine-depleted medium invitro and severely compromised the invivo growth of tumor xenografts. These results suggest that the lack of asparaginase activity represents an evolutionary adaptation to allow mammalian cells to survive pathophysiologic variations in extracellular glutamine.

Abstract 30: Kras mediates amino acid metabolism during nutrient stress

Abstract Kras is a small GTPase that functions in the transmission of extracellular mitogenic stimuli, and is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC). Previously, Kras has been demonstrated to play a role in tumor metabolism in certain cell types and cancers, but this has yet to be analyzed in the context of NSCLC. We have analyzed how Kras changes metabolic gene expression by expressing a doxycycline-inducible hairpin against Kras in 4 NSCLC cell lines. One of the most regulated metabolism genes is asparagine synthetase (Asns), which transfers the γ-amino group of glutamine to aspartate, yielding glutamate and asparagine. We find elevated levels of Asns in Kras mutant tumors, and that expression of Asns correlates with poor patient outcome. Knockdown of Asns in the absence of exogenous asparagine in the media results in inhibition of cell growth and induction of apoptosis that is rescued by addition of asparagine. Additionally, under conditions of low glutamine levels, knockdown of Asns sensitizes cells to apoptosis, and overexpression of Asns is protective under these conditions. We also find a role for the glutaminase-activity of Asns in glutamine-mediated anapleurosis of the TCA cycle. These observations suggest Asns may be an interesting therapeutic target for NSCLC tumors with mutations in Kras. Citation Format: Dana Gwinn, Alejandro Sweet-Cordero. Kras mediates amino acid metabolism during nutrient stress. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 30.

Oncogenic KRAS regulates asparagine synthase

Kras is a small GTPase that functions in the transmission of extracellular mitogenic stimuli, and is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC). Previously, Kras has been demonstrated to play a role in tumor metabolism in certain cell types and cancers, but this has yet to be analyzed in the context of NSCLC. We have analyzed how Kras changes metabolic gene expression by expressing a doxycycline-inducible hairpin against Kras in several NSCLC cell lines. One of the most regulated metabolism genes is asparagine synthetase (Asns), which transfers the g-amino group of glutamine to aspartate, yielding glutamate and asparagine. We find elevated levels of Asns in Kras mutant tumors, and that expression of Asns correlates with poor patient outcome. Knockdown of Asns in the absence of exogenous asparagine in the media results in inhibition of cell growth and induction of apoptosis that is rescued by addition of asparagine. Additionally, under conditions of low glutamine levels, knockdown of Asns sensitizes cells to apoptosis, and overexpression of Asns is protective under these conditions. We also find a role for the glutaminase-activity of Asns in glutaminemediated anapleurosis of the TCA cycle. These observations suggest Asns may be an interesting and novel therapeutic target for NSCLC tumors with mutations in Kras.

Abstract B27: Kras alters expression of asparagine synthetase (Asns) in non-small cell lung cancer (NSCLC) and protects tumors during nutrient stress

Abstract Kras is a small GTPase that functions in the transmission of extracellular mitogenic stimuli, and is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC). Previously, Kras has been demonstrated to play a role in tumor metabolism in certain cell types and cancers, but this has yet to be analyzed in the context of NSCLC. We have analyzed how Kras changes metabolic gene expression by expressing a doxycycline-inducible hairpin against Kras in 4 NSCLC cell lines. One of the most regulated metabolism genes is asparagine synthetase (Asns), which transfers the γ-amino group of glutamine to aspartate, yielding glutamate and asparagine. We find elevated levels of Asns in Kras mutant tumors, and that expression of Asns correlates with poor patient outcome. Knockdown of Asns in the absence of exogenous asparagine in the media results in inhibition of cell growth and induction of apoptosis that is rescued by addition of asparagine. Additionally, under conditions of low glutamine levels, knockdown of Asns sensitizes cells to apoptosis, and overexpression of Asns is protective under these conditions. We also find a role for the glutaminase-activity of Asns in glutamine-mediated anapleurosis of the TCA cycle. These observations suggest Asns may be an interesting therapeutic target for NSCLC tumors with mutations in Kras. Citation Format: Dana M. Gwinn, Alejandro Sweet-Cordero. Kras alters expression of asparagine synthetase (Asns) in non-small cell lung cancer (NSCLC) and protects tumors during nutrient stress. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B27.

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.

Acute metabolic encephalopathy in two patients treated with asparaginase and ondasetron

L-asparaginase has been an important agent of acute lymphoblastic leukemia (ALL) therapy for more than 40 years. The major adverse events of L-asparaginase are immunoallergic reaction, pancreatitis, liver cytolysis (increased ASAT, ALAT, and bilirubin), and cerebral haemorrhage or thrombosis. Hyperammonemia has already been described with L -asparaginase treatment [1]. In the present article, we report acute metabolic encephalopathy in two young boys receiving asparaginase for B ALL relapse. We discuss the pathophysiology of this metabolic encephalopathy with hyperammonemia and its medical management.

The Inhibition of Lymphocyte Blastogenesis by Asparaginase: Critical Role of Glutamine in both T and B Lymphocyte Transformation

L-Asparaginase has long been used in the treatment of acute lymphoblastic leukemia or malignant lymphoma in childhood. To determine cell type specific sensitivity to this drug, the L-asparaginase-mediated inhibition of blastogenesis of human peripheral T or B lymphocytes was compared. The rate of incorporation of [3H]-thymidine into the DNA of either T lymphocytes due to phytohemagglutinin (PHA) or B lymphocytes due to Staphylococcus aureus Cowan I (SAC) was measured by the addition of Escherichia coli L-asparaginase in the medium. The blastogenic response of either T or B lymphocytes was also determined in medium depleted of exogenous asparagine and/or glutamine, both of which are hydrolyzed by this enzyme. The in vitro blastogenesis of either human T lymphocytes due to PHA or B lymphocytes due to SAC was inhibited by the inclusion of asparaginase in the medium. The deprivation of exogenous asparagine did not have any inhibitory effect on the blastogenic response of both T and B lymphocytes to each mitogen. On the other hand, the glutamine concentration in the culture medium provided a critical influence on the proliferative response of T and B lymphocytes. The rate of incorporation of [3H]-thymidine into DNA was increased markedly as the concentration of glutamine was increased from 2(-7)-2 mmol/l. It is concluded that the mechanism of inhibition of PHA- or SAC-stimulated lymphocyte blastogenesis by L-asparaginase is not asparagine deprivation but glutamine deprivation. Glutamine, which is the most abundant amino acid, is thought to have an important role in the immune response of lymphocytes.

Organization and expression of the cell cycle gene, ts11, that encodes asparagine synthetase.

The human ts11 gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle ts11 mutant, which is blocked in G1 at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the ts11 block. The ts11 (AS) mRNA has a size of about 2 kilobases and is induced in mid-G1 phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the ts11 gene. The human ts11 gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts11 P1) contained in a 280-base-pair region upstream of the first exon and the other (ts11 P2) contained in the first intron. ts11 P1 produced about sixfold more chloramphenicol acetyltransferase activity than did ts11 P2 and had features of the promoters of housekeeping genes: high G + C content, multiple transcription start sites, absence of a TATA box, and presence of putative Sp1 binding sites. ts11 P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The ts11 gene was overexpressed in ts11 cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented G1 induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.

Organization and Expression of the Cell Cycle Gene, ts11, That Encodes Asparagine Synthetase

The human ts11 gene was isolated on the basis of its ability to complement the mutation of the BHK cell cycle ts11 mutant, which is blocked in G1 at the nonpermissive temperature. This gene has now been identified as the structural gene for asparagine synthetase (AS) on the bases of sequence homology and the ability of exogenous asparagine to bypass the ts11 block. The ts11 (AS) mRNA has a size of about 2 kilobases and is induced in mid-G1 phase in human, mouse, and hamster cell lines. We have studied the organization and regulation of expression of the ts11 gene. The human ts11 gene consists of 13 exons (the first two noncoding) interspersed in a region of about 21 kilobases of DNA. Transient expression assays using the bacterial chloramphenicol acetyltransferase reporter gene identified two separate promoters: one (ts11 P1) contained in a 280-base-pair region upstream of the first exon and the other (ts11 P2) contained in the first intron. ts11 P1 produced about sixfold more chloramphenicol acetyltransferase activity than did ts11 P2 and had features of the promoters of housekeeping genes: high G + C content, multiple transcription start sites, absence of a TATA box, and presence of putative Sp1 binding sites. ts11 P2 contained a TATA sequence and other elements characteristic of a promoter, but so far we have no evidence of its physiological utilization. The ts11 gene was overexpressed in ts11 cells exposed to the nonpermissive temperature. Addition of asparagine to the culture medium led to a drastic decrease in mRNA levels and prevented G1 induction in serum-stimulated cells, which indicated that expression of the AS gene is regulated by a mechanism of end product inhibition.

The stimulus-secretion coupling of amino acid-induced insulin release metabolic interaction L-asparagine and L-leucine in pancreatic islets

1. Because L-asparagine augments insulin release evoked by L-leucine, the metabolism of these two amino acids was investigated in rat pancreatic islets. 2. L-Leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert any obvious primary effect upon the further catabolism of aspartate derived from exogenous asparagine. 3. L-Asparagine augmented the oxidation of L-leucine, and effect possibly attributable to activaion of 2-ketoisocaproate dehydrogenase. 4. The association of L-asparagine and L-leucine exerted a sparing action on the utilization of endogenous amino acids, so that the integrated rate of nutrients oxidation was virtually identical in the sole presence of L-leucine and simultaneous presence of L-asparagine and L-leucine, respectively. 5. It is proposed that the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attributable to an increased generation rate of cytosolic NADPH rather than any increase in nutrients oxidation.

Amphibian cells in culture. I. Nutritional studies

Nutritional requirements of amphibian cells in culture were studied for the purpose of modifying a minimal medium in which frog cells could proliferate and which could be used for obtaining drug-resistant and auxotrophic variants. The serum, purine, CO2, and amino acid requirements for ICR 2A (a Rana pipiens haploid cell strain) have been investigated employing two different media: L-15, a nonbicarbonate, amino acid-buffered medium and Eagle's MEM, a bicarbonate-buffered medium. In this paper we present evidence to support the following conclusions: (1) With L-15 as the base medium, 10% fetal calf serum (FCS) supports optimal cell growth during exponential phase. Calf serum, whole, dialyzed, or heat-inactivated, cannot substitute for FCS and, in fact, is inhibitory. (2) Purines are required by ICR 2A cells only if grown in a nonbicarbonate-buffered medium, since the cells under these conditions cannot produce enough endogenous CO2 to support de novo purine synthesis. (3) In addition to the amino acids considered essential for mammalian cells in culture, ICR 2A cells depend upon exogenous asparagine. Glutamine and/or aspartic acid cannot replace the asparagine requirement. However, ICR 2A cells do utilized exogenous glutamine as an oxidative substrate.