Reply to Stams et al

Krejčí, Ondřej; Starková, Júlia; Otová, B; Madžo, Jozef; Kalinová, Markéta; Hrušák, Ondřej; Trka, Jan

doi:10.1038/sj.leu.2403577

Cited by 3 publications

(4 citation statements)

References 6 publications

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“…Our results contradict those reported by both Stams et al (2003) and Krejci et al (2004) in that AS mRNA levels in NPBMC were found to be significantly higher than those in lymphoblasts, in line with the expectation from many in vitro studies. As previously discussed (Krejci et al , 2005a,b, Stams et al , 2005) this may be due to the choice of the endogenous control used to normalise AS expression. We selected TBP as a suitable control as the variation of TBP expression between samples of lymphoid origin has been shown to be lower than either GAPDH or β 2microglobulin (Lossos et al , 2003) and, in our RQ RT‐PCR assay, the abundance of TBP was similar to that of AS .…”

Section: Resultsmentioning

confidence: 99%

Expression levels of asparagine synthetase in blasts from children and adults with acute lymphoblastic leukaemia

et al. 2006

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l-asparaginase (l-asp) is an important component in the treatment of childhood acute lymphoblastic leukaemia (ALL).Recently the assumption that leukaemic cells are sensitive to l-asp because of a relative lack of asparagine synthetase (AS) has been questioned (Stams et al, 2003;Krejci et al, 2004). Using a real-time quantitative reverse transcription polymerase chain reaction (RQ RT-PCR) assay with transcription binding protein IID/TATA (TBP) as the internal control we provide evidence to support the original supposition that lymphoblasts have significantly lower levels of expression of AS than normal peripheral blood mononuclear cells (NPBMC). Materials and methodsFollowing ethical approval, diagnostic bone marrow samples were obtained from 59 children (median 5 years; range 0AE6-15AE5 years) and 23 adults (median 29 years; range 17AE7-71 years) with ALL and eight children (median 5AE2 years; range 1-16 years) and 22 adults (median 57; range 17-84 years) with acute myeloid leukaemia (AML). The presence of >80% blasts were confirmed by morphology. NPBMC were obtained from 14 healthy volunteers. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep (Nycomed Pharma, Oslo, Norway). K562, MOLT-4 and NALM-6 cell lines were grown under standard conditions for use as controls.Total mRNA was extracted using the RNeasy mini kit (Qiagen, Crawley, UK) according to the manufacturer's protocol. Reverse transcription of 200 ng total RNA was performed using a Taqman reverse transcription kit (Applied Biosystems, Warrington, UK) with random hexomers. Primers and Taqman probe for AS RQ RT-PCR, were designed using Primer-Express (Applied Biosystems). Primer and probe sequences were as follows; forward -5¢-TCAGCCCGCCACAT-CAC-3¢-(in exon 1), reverse -5¢-CAATGAAGCTATAAG-CTTTCTTCAAGTG-3¢ (spanning exon 2 and 3), probe, -5¢-CTGACCTGCTTACGCCCAGATTTTCTTCAA-3¢ (spanning exon one and two). Primers and probe for TBP were purchased as a Taqman pre-developed control reagent.Standards containing 75, 18AE75, 4AE69, 1AE17, 0AE29 and 0AE07 ng of K562 total RNA with reverse transcribed Escherichia coli tRNA as a carrier were assayed for AS and TBP in triplicate on 27 separate occasions. Computerised tomography (CT) values (the fractional cycle numbers at which the fluorescence passed the fixed threshold) for each standard were obtained using an ABI Prism 5700 sequence detection system (Applied Biosystems, Warrington, UK) and the average CT for each point plotted against log standard RNA concentration.Reaction mixtures (82AE5 ll) contained 41AE25 ll Taqman PCR universal master mix, 900 nmol/l AS forward primer, 900 nmol/l AS reverse primer, 225 nmol/l AS probe or 4AE13 ll of ready synthesised TBP primers and probe and either 7AE3 ll of sample cDNA, 22AE5 ll of standard cDNA or 3AE2 ll of calibrator cDNA. Aliquots (25 ll) were added to wells of a microplate in triplicate. The thermal cycling conditions were 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min.A calibrator sample was quantified for AS and TBP ...

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Section: Resultsmentioning

confidence: 99%

Expression levels of asparagine synthetase in blasts from children and adults with acute lymphoblastic leukaemia

et al. 2006

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“…l ‐Asparaginase ( l ‐Asnase) obtained from Escherichia coli is a kind of enzyme that can hydrolyze l ‐asparagine ( l ‐Asn) and l ‐glutamine ( l ‐Gln) to l ‐aspartic acid and l ‐glutamic acid ( l ‐Glu), respectively. Due to its rapid depletion of l ‐Asn and suppression effect in the growth of malignant cells, l ‐Asnase can be employed as an important drug for the treatment of acute lymphoblastic leukemia (ALL) . However, the control efficacy of l ‐Asnase is limited due to its cytotoxic effect and the secondary complications, including coagulation abnormalities, hepatic, and pancreatic dysfunction .…”

Section: Introductionmentioning

confidence: 99%

Microchip CE‐LIF method for the hydrolysis of L‐glutamine by using L‐asparaginase enzyme reactor based on gold nanoparticle

Qiao

Yan

et al. 2013

Electrophoresis

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L-Asparaginase (L-Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, L-glutamine (L-Gln) and L-asparagine (L-Asn). To study the cytotoxic effect and the secondary complications of L-Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of L-Asnase for the hydrolysis of L-Gln, employing the enzyme-gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)-LIF was established for the separation of L-amino acids (L-Gln and L-glutamic acid) and studying the hydrolysis of L-Gln by using L-Asnase enzyme reactor. Then, using L-Gln as target analyte, the enzyme kinetics of L-Asnase in free solution, enzyme-gold nanoparticle conjugates (E-GNP), and the enzyme-gold nanoparticle conjugates immobilized in capillary (E-GNP-C) were investigated in detail with the proposed MCE-LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of L-Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E-GNP-C enzyme reactor exhibited the best performance for the hydrolysis of L-Gln.

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“…Since 1967, L-asparaginase (L-Asnase) has been used as an integral part of the treatment of child ALL. 1 It can catalyze the hydrolysis of L-asparagine (L-Asn) to L-aspartic acid (L-Asp) and ammonia. Thus, the relative deficiency of L-Asn in the serum and endogenous L-Asn synthesis could cause the apoptosis and impair protein synthesis in leukemic blasts.…”

Section: Introductionmentioning

confidence: 99%

“…2 Results showed that L-Asnase could cause complete remission in 40-60% of paediatric ALL cases when it was used alone. 1 Although L-Asnase is effective in the treatment of malignant diseases, many side effects, including hepatic toxicity, allergic reaction, pancreatitis, central nervous system toxicity and decreased synthesis of blood clotting factors, have been reported about this standard chemotherapy component for ALL. 3 It was well documented that L-Asnase was the leading cause of hypersensitivity reactions in 5 to 45% of the patients 4 and thrombotic events varying from 1.1 to 73% in children.…”

Section: Introductionmentioning

confidence: 99%

Monolith and coating enzymatic microreactors of l-asparaginase: kinetics study by MCE–LIF for potential application in acute lymphoblastic leukemia (ALL) treatment

Qiao

et al. 2011

Analyst

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The study of enzyme immobilization using an extracorporeal shunt system is essential to eliminate the side effects of L-asparaginase (L-Asnase; including hepatic toxicity, allergic reaction, pancreatitis, central nervous system toxicity and decreased synthesis of blood clotting factors) when it was applied as an anticancer drug given directly to patients by injection. Thus, the novel monolith and coating enzymatic reactors of L-asparaginase were provided in this assay and a microchip electrophoresis-laser induced fluorescence (MCE-LIF) method was set up for the enzyme kinetics study. The enzymatic reactors would be a promising in vitro therapeutic method in an extracorporeal shunt system for acute lymphoblastic leukemia (ALL) treatment. For the first time, L-asparaginase was covalently bound to the polymer monolith and coating in the capillary and the activity characteristics of these enzymatic microreactors have been probed by Michaelis-Menten kinetic constants. Meanwhile, the D,L-amino acids were chirally separated using microchip electrophoresis with a laser induced detector and D,L-aspartic acid (D,L-Asp) were tested for the L-asparaginase enzymatic reactor kinetics study. Furthermore, human serum adding with L-asparagine (L-Asn) as the sample was hydrolyzed by the enzymatic microreactors. The results demonstrated that the developed enzymatic microreactor of L-asparaginase would be a potential therapeutic protocol for ALL treatment.

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Reply to Stams et al

Cited by 3 publications

References 6 publications

Expression levels of asparagine synthetase in blasts from children and adults with acute lymphoblastic leukaemia

Expression levels of asparagine synthetase in blasts from children and adults with acute lymphoblastic leukaemia

Microchip CE‐LIF method for the hydrolysis of L‐glutamine by using L‐asparaginase enzyme reactor based on gold nanoparticle

Monolith and coating enzymatic microreactors of l-asparaginase: kinetics study by MCE–LIF for potential application in acute lymphoblastic leukemia (ALL) treatment

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