Haematologica, Vol 94, Issue 9, 1322-1324 doi:10.3324/haematol.2009.011437
Copyright © 2009 by Ferrata Storti Foundation
Esther Hulleman ,
Mathilde JC Broekhuis ,
Rob Pieters ,
Monique L Den Boer
Next, the expression of the different PK isoforms was determined in normal peripheral blood lymphocytes, normal bone marrow and in leukemic samples from untreated children at initial diagnosis of ALL that were identified by an in vitro cytotoxicity assay (MTT)8 as prednisolone-resistant (n=11, LC50
150 µg/mL), or sensitive to prednisolone (n=19, LC50
0.1 µg/mL).6 In correspondence with the results of Christofk et al.,7 a significant difference (p<0.0001) was found in the expression of PKM2 between normal and ALL cells (Figure 2A). However, PKM2 transcripts were found in all patient samples and no significant difference was observed between prednisolone-resistant or prednisolone-sensitive ALL cases (Figure 2B). Similar expression patterns were observed for PKM1/2 (Figure 2C and D), although the transcript levels of PKM1/2 were about ten-fold higher than the expression levels of PKM2. Together, these data indicate that not only the M2 isoform of pyruvate kinase was present in ALL patients but also PKM1, and that the mRNA expression levels of different PKM isoforms are no indication of glucocorticoid resistance in childhood leukemia.
Since it has been reported that tumor cells exclusively express the M2 isoform of pyruvate kinase7,9–11 and not PKM1, we also determined the expression of the different PKM isoforms at the protein level. Western blotting was performed using antibodies that, like the primers in the Q-PCR experiments, specifically recognized PKM2 or both PKM1/2 isoforms. Blots incubated with these antibodies showed a clear band for PKM1/2, but not PKM2, in normal lymphocytes (Figure 2E). In samples from ALL patients, however, equivalent expression levels of PKM1/2 and PKM2 were present (Figure 2F). Analogous to the Q-PCR results, no significant difference in protein levels were observed between prednisolone-resistant and prednisolone-sensitive cases, suggesting that pyruvate kinase isoform M2 is not responsible for glucocorticoid resistance in childhood leukemia. Whether the difference in expression between different isoforms of pyruvate kinase that was detected between normal bone marrow and leukemic cells reflects a difference in glycolytic rate is not known, since patient cells do not grow in vitro and we can not detect glucose consumption. Thus, although pyruvate kinase might play a role in the regulation of glycolysis in childhood ALL, glucocorticoid resistance is unlikely to be caused by selective expression of PKM2. 
Funding: this work was financially supported by the Dutch Cancer Society (grant EMCR 2005-3313).
Copyright © 2009 by Ferrata Storti Foundation
Acute Lymphobastic Leukemia |
Pyruvate kinase M2 and prednisolone resistance in acute lymphoblastic leukemia
Erasmus MC - Sophia Children’s Hospital, University Medical Center, Dept. of Pediatric Oncology and Hematology, Rotterdam, The Netherlands
Correspondence: Monique L. den Boer, Erasmus MC, Sophia Children’s Hospital, Dept. of Pediatric Oncology and Hematology, Dr Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands, Phone: international +31.10.7036691. Fax: international +31.10.7044761. E-mail:m.l.denboer{at}erasmusmc.nl
Key words: pyruvate kinase, glycolysis, perdnisolone, resistance, childhood ALL.
Treatment of childhood acute lymphoblastic leukemia (ALL) combines different classes of chemotherapeutic agents, such as Vinca alkaloids, anthracyclines and glucocorticoids (GCs). Although such therapy nowadays cures the majority of the patients, combination chemotherapy still fails in approximately 20%.1 Most treatment failures can be explained by resistance to antileukemic agents,2 and resistance to the glucocorticoids in particular has been shown to be related to an unfavorable event free survival.3,4 Since the glucocorticoids prednisolone and dexamethasone play a crucial role in essentially all therapy protocols, the development of strategies to reverse resistance to these agents is important to improve ALL treatment efficacy. Recently, we have demonstrated that glucocorticoid resistance in pediatric ALL is associated with increased glucose metabolism, and that inhibition of glycolysis sensitizes prednisolone-resistant ALL cells to glucocorticoids.5 Although it has been known for several decades that cancer cells shift their energy production from oxidative phosphorylation towards the less efficient glycolysis pathway (the so called Warburg effect)6 it is still not clear how tumor cells establish this altered metabolic phenotype. Christofk et al. recently showed that altered expression of the glycolytic enzyme pyruvate kinase (PK), and more specifically the switch to the alternatively spliced isoform M2 (PKM2), is responsible for the increased rate of glycolysis observed in cancer cells.7 Together, these findings suggest an upregulation of PKM2 in prednisolone resistant leukemia.
To investigate if pyruvate kinase M2 plays a role in glucocorticoid resistance in pediatric ALL, the expression of PKM2 was determined in leukemic cell samples of ALL patients and compared to PKM2 expression in normal peripheral blood and bone marrow samples. To distinguish between the different isoforms in real time quantitative PCR (RT Q-PCR), primers were designed that specifically amplify PKM2 or that recognize both isoform 1 and 2 of pyruvate kinase (PKM1/2, Figure 1A). The expression of PK isoforms was subsequently tested in different cell lines, confirming the specificity of the primer combinations (Figure 1B).
 View larger version (30K): [in a new window] [Download PPT slide] |
Figure 1. Verification of the real time PCR primer-probe specificity. (A) Graphic representation of primer design to distinguish pyruvate kinase isoforms. The primers located in exon 3 can not distinguish different PKM isoforms (PKM1/2). A primer-probe combination amplifying nt 1586–1692 (exon 9) was used to specifically amplify PKM2. Amplification efficiency was over 95% for all primer-probe combinations. Ribosomal protein S20 (RPS20) was used as a control gene for normalization. (B) Graphic representation of mRNA levels of pyruvate kinase isoform M2 (left panel) or isoform 1 and 2 (right panel) in different leukemic cell lines confirming primer-probe specificity. Expression of PK-M1/2 in Jurkat cells was set to be 100% and relative expression levels were calculated.
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150 µg/mL), or sensitive to prednisolone (n=19, LC500.1 µg/mL).6 In correspondence with the results of Christofk et al.,7 a significant difference (p<0.0001) was found in the expression of PKM2 between normal and ALL cells (Figure 2A). However, PKM2 transcripts were found in all patient samples and no significant difference was observed between prednisolone-resistant or prednisolone-sensitive ALL cases (Figure 2B). Similar expression patterns were observed for PKM1/2 (Figure 2C and D), although the transcript levels of PKM1/2 were about ten-fold higher than the expression levels of PKM2. Together, these data indicate that not only the M2 isoform of pyruvate kinase was present in ALL patients but also PKM1, and that the mRNA expression levels of different PKM isoforms are no indication of glucocorticoid resistance in childhood leukemia.
 View larger version (20K): [in a new window] [Download PPT slide] |
Figure 2. Expression of PKM isoforms in ALL patients. (A–D) Graphical representation of the expression of pyruvate kinase isoform M2 (panels a, b) or isoform M1 and M2 (panels c, d) as measured by Q-PCR. Expression of PK isoforms in normal lymphocytes versus ALL cells is depicted in panels a and c; expression of PK isoforms in prednisolone-resistant or prednisolone–sensitive patients in panels b and d. As normal samples peripheral blood ( ) or bone marrow ( ) was used. Medians are indicated as horizontal lines. (E–F) Western blots representing the expression of pyruvate kinase isoforms in normal lymphocytes (panel e) or in prednisolone-resistant or prednisolone–sensitive patients (panel f). 20 µg of protein was loaded and membranes were incubated with 1:1000 diluted antibody directed against PKM1/2 or PKM2 (Cell Signaling Technology Inc., Danvers USA).
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Footnotes
Funding: this work was financially supported by the Dutch Cancer Society (grant EMCR 2005-3313).
References
 TOP References |
|---|
- Pui CH, Evans WE. Treatment of acute lymphoblastic leukemia. N Engl J Med 2006;354:166-78.
[Free Full Text] - Pieters R, Klumper E, Kaspers GJ, Veerman AJ. Everything you always wanted to know about cellular drug resistance in childhood acute lymphoblastic leukemia. Crit Rev Oncol Hematol 1997;25:11-26.[Web of Science][Medline]
- Kaspers GJ, Pieters R, Van Zantwijk CH, Van Wering ER, Van Der Does-Van den Berg A, Veerman AJ. Prednisolone resistance in childhood acute lymphoblastic leukemia: vitro-vivo correlations and cross-resistance to other drugs. Blood 1998;92:259-66.
[Abstract/Free Full Text] - Den Boer ML, Harms DO, Pieters R, Kazemier KM, Gobel U, Körholz D, et al. Patient stratification based on prednisolone-vincristine-asparaginase resistance profiles in children with acute lymphoblastic leukemia. J Clin Oncol 2003;21:3262-8.
[Abstract/Free Full Text] - Hulleman E, Kazemier KM, Holleman A, Vanderweele DJ, Rudin CM, Broekhuis MJ, et al. Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells. Blood 2009;113:2014-21.
[Abstract/Free Full Text] - Kim JW, Dang CV. Cancer’s molecular sweet tooth and the Warburg effect. Cancer Res 2006;66:8927-30.
[Abstract/Free Full Text] - Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A, Gerszten RE, Wei R, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 2008;452:230-3.[CrossRef][Web of Science][Medline]
- Pieters R, Loonen AH, Huismans DR, Broekema GJ, Dirven MW, Heyenbrok MW, et al. In vitro drug sensitivity of cells from children with leukemia using the MTT assay with improved culture conditions. Blood 1990;76:2327-36.
[Abstract/Free Full Text] - Mazurek S, Boschek CB, Hugo F, Eigenbrodt E. Pyruvate kinase type M2 and its role in tumor growth and spreading. Semin Cancer Biol 2005;15:300-8.[CrossRef][Web of Science][Medline]
- Dombrauckas JD, Santarsiero BD, Mesecar AD. Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis. Biochemistry 2005;44:9417-29.[CrossRef][Web of Science][Medline]
- Koss K, Harrison RF, Gregory J, Darnton SJ, Anderson MR, Jankwski JA. The metabolic marker tumour pyruvate kinase type M2 (tumour M2-PK) shows increased expression along the metaplasia-dysplasia-adenocarcinoma sequence in Barrett’s oesophagus. J Clin Pathol 2004;57:1156-9.
[Abstract/Free Full Text]
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