Leukemia (2010) 24, 1513–1541 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu LETTERS TO THE EDITOR

Deoxycytidine is downregulated in Ara-C-resistant acute myeloid leukemia murine cell lines

Leukemia (2010) 24, 1513–1515; doi:10.1038/leu.2010.88; different passages) using Affymetrix (Santa Clara, CA, USA) published online 27 May 2010 Mouse Genome 430 2.0 Arrays containing 45 101 murine oligonucleotide sequences. The detection values were pro- Acute myeloid leukemia (AML) is a malignant expansion of cessed using Genedata Expressionist software (Genedata AG, immature cells in the bone marrow. Although chemotherapeutic Basel, Switzerland) using the robust multiarray average normal- treatments containing cytosine arabinoside (Ara-C) typically ization technique. The raw data are available online at http:// result in dramatic remissions, resistance to www.ncbi.nlm.nih.gov/geo/ (accession number GSE18322). frequently occurs. Patients presenting with Ara-C-resistant Group t-test analysis was used to compare the B117P cells to AML have a reduction in the active metabolized form of the B117H cells, and the B140P cells to the B140H cells. Only Ara-C (Ara-CTP), but the mechanisms responsible for this expression changes greater than 2.0-fold with P-values o0.001 reduction are still being investigated. In previously published were considered. One hundred and twenty differentially work, two AML cell lines (B117P and B140P), derived from expressed probe sets representing 104 were obtained BXH-2 strain mouse leukemias, were subjected to increasing when comparing B140H to B140P. Two hundred and eighty six concentrations of Ara-C to create two highly resistant derivatives differentially expressed probe sets representing 239 genes were (B117H and B140H) that tolerate Ara-C concentrations B800 obtained when comparing B117H to B117P. In Table 1 those times that of their parental lines.1 In this study, expression genes with consistent expression changes (42.0-fold) on microarrays were conducted to determine the expression selection for Ara-C resistance in both cell lines are listed. changes common to both sets of cell lines. The results were The kinase (Dck) gene mRNA level was altered verified by quantitative PCR (qPCR) and the specificity of drug to a greater extent than any other single gene. Starting levels of resistance was evaluated. Dck expression were similar in both the B117P and B140P The parental cell lines (B117P and B140P) are markedly parental cell lines. However, the Dck levels in the B117H cells different phenotypically and yet their response to Ara-C is about were near zero, whereas the Dck levels in B140H cells were the same with similar 50% inhibitory concentrations (IC50). decreased 8.0-fold compared to B140P. The probe sets used to B117P grows 2.3 times faster than B140P (data not shown), and measure Dck expression targeted the 30-UTR of Dck. Expression they have differences in patterns, as detected by changes for the Dck gene were confirmed by qPCR analysis microarray analysis. Although CD34 has not been found to be using the Eppendorf (Hamburg, Germany) Mastercycler ep associated with any particular AML subtype or to be prognos- realplex. PCR primers were designed to target the 30 end of tically significant, B117P and B117H highly express Cd34 the Dck transcript. Rps9, a gene that is expressed at moderate whereas B140P and B140H do not (a 183-fold difference). and consistent levels across all four cell lines, was used to Furthermore, the level of myeloperoxidase (Mpo) is 34-fold normalize the results. The qPCR analysis confirmed the altered greater in the B117P cells when compared with the B140P cells. expression levels found in the microarray (Figure 1). DCK Despite these differences, both cell lines showed some common encodes deoxycytidine kinase, which phosphorylates deoxyr- changes in gene expression on selection for Ara-C resistance. ibonucleosides including deoxycytidine, and Microarray gene expression analysis to compare differences . DCK performs the rate-limiting step in Ara-C between the parental cell lines (B117P and B140P) and their metabolic activation, which is monophosphosphorylation after highly resistant derivatives (B117H and B140H) was performed import of Ara-C through the SLC29A1 nucleoside transporter. on 12 RNA isolations (3 isolations for each cell line from Thus, downregulation of this kinase would be expected to lead

Table 1 Common gene expression changes between Ara-C-resistant cell lines and their Ara-C-sensitive parental lines (42.0-fold)

Probe ID Gene ID Description Fold change

B117H vs B117P B140H vs B140P

1439012_a_at Dck Deoxycytidine kinase À273.157 À8.264 1449176_a_at Dck Deoxycytidine kinase À92.503 À7.488 1415673_at Psph Phosphoserine phosphatase À4.571 À2.606 1424254_at Ifitm1 Interferon-induced transmembrane protein 1 À4.456 À2.788 1420498_a_at Dab2 Disabled homolog 2 (Drosophila) À4.338 À3.251 1423805_at Dab2 Disabled homolog 2 (Drosophila) À3.632 À2.108 1429693_at Dab2 Disabled homolog 2 (Drosophila) À3.566 À2.480 1455991_at Ccbl2 Cysteine conjugate-b lyase 2 À2.176 À3.308 1433521_at Ankrd13c Ankyrin repeat domain 13C 2.055 2.021 1428114_at Slc14a1 Solute carrier family 14 (urea transporter), member 1 2.692 2.135 1433939_at Aff3 AF4/FMR2 family, member 3 2.737 2.180 Letters to the Editor 1514 to resistance to Ara-C. Indeed, polymorphisms in the human Manipulation of the Wnt pathway may also be involved in DCK gene have been associated with patient’s response to AML Ara-C resistance. Dab2, an adaptor protein that stabilizes containing Ara-C,2 and expression of alterna- Axin, and thus blocks Wnt signaling,5 is downregulated in tively spliced isoforms of DCK are found in some Ara-C-resistant both Ara-C-resistant cell lines, suggesting an increase of Wnt/b- AML patients.3 A recent paper described alterations in DCK catenin signaling may be present in the Ara-C-resistant expression in a human AML cell line selected for resistance to derivatives. Ara-C in vitro.4 These investigators also noted that primary Another gene of particular interest is Ifitm1, which was human AML samples differ in the level of DCK transcript, and significantly downregulated in both Ara-C-resistant cell lines. that these differences may relate to Ara-C sensitivity, at least as Ifitm1, also called Leu13, is an interferon-induced antiprolifera- measured in an in vitro assay. tive transmembrane protein. Lower levels of IFITM1 expression Drug assays were conducted on all four cell lines to determine have been shown to correlate with decreased survival in chronic if Ara-C resistance conferred drug resistance to other chemo- myeloid leukemia.6 A decrease in IFITM1 expression in therapy drugs. There were no changes in response to dauno- esophageal squamous cell carcinoma has been shown to rubicin or etoposide (data not shown). However, there was a correlate with increased resistance to cis-platinum. significant change in the response to decitabine in both sets of Because the gene expression patterns of B117P and B140P cells, with a greater than 800-fold increase in IC50 values in Ara-C- cells are so different, analysis of gene expression changes for resistant derivatives compared to parental cell lines (Figure 2). each cell line on selection for Ara-C may provide clues Both Ara-C and decitabine are deoxycytidine analogs, and are for identifying additional drug resistance genes (Tables 2 triphosphorylated by the same (including Dck). How- and 3). For example, in the B140H cells, there was a 36-fold ever, their methods of interfering with cell proliferation are downregulation of Spint2, which has been characterized as a entirely different. Once triphosphorylated, Ara-C is incorporated tumor suppressor gene in medulloblastoma.7 B117H cells have into DNA during replication, blocking and topoi- upregulated the neuregulin-4 (Nrg4) gene transcript 10-fold. somerase activity, whereas decitabine inhibits DNA methyl- Nrg4 can bind to and activate epidermal growth factor receptor activity. Because the change in resistance to both family receptors and so their activation could contribute to the Ara-C and decitabine are comparable, this supports the Ara-C-resistant phenotype in this cell line perhaps by activation hypothesis that the Ara-C drug resistance of the B117H and of the RAS/MAPK pathway that has been shown to modulate B140H cells is driven specifically by the downregulation of Dck. Ara-C responsiveness. S100a10 gene expression is dramatically However, there are other changes taking place in the cells downregulated in B140H cells, and the product of this gene that may have a supporting role in the development of drug interacts with Annexin-11, which has been described as an resistance. For example, the urea transporter Slc14a1 is anti-apoptotic protein involved in chemotherapy resistance.8 upregulated in both sets of Ara-C-resistant cells. This suggests Further studies are underway to determine the mechanisms that the Ara-C-resistant cells are producing more urea than their behind the reduction in Dck expression and to identify whether Ara-C-sensitive parental cells. Downregulation of Dck would the expression changes of other genes identified by the also interfere with the cells ability to get deoxycytidines through microarray analysis are required by the cells to compensate the salvage pathway. So, one possible explanation for this for loss of Dck expression or participate in the development of increase in urea is that the Ara-C-resistant cells are deriving the Ara-C resistance by other mechanisms. Drugs targeting the building blocks for de novo synthesis of cytosine from the pathways that can cooperate with Dck downregulation to allow degradation of thymidine. Urea is a by-product of the thymidine Ara-C resistance may be useful for preventing the selection of degradation process. Ara-C-resistant clones in patients.

qPCR Measurement of Dck Expression Response of Cells to Decitabine 0.03 3 M) Rps9 2 µ µ ( to 50 0.02 Dck 1

0

0.01 -1 Log of Decitabine IC

Relative expresion of -2 B117PB117H B140P B140H 0 B117PB117H B140P B140H Figure 2 Response of Ara-C-resistant cell lines and their parental lines to decitabine. CellTiter 96 AQueous Non-Radioactive Cell Proli- Figure 1 qPCR measurement of Dck expression in Ara-C-resistant feration Assay (Promega Corp, Madison, WI, USA) was used to cell lines and their parental lines. qPCR was repeated three times for measure inhibitory concentration using previously described meth- 1 each cell line. Rps9 was used to normalize the data. Primer pairs used ods. Each cell line was evaluated three or more times. IC50 was for Dck were 50-TGAGGATAGAACATGCCAAGG-30 and 50-CG calculated using CalcuSyn software (Biosoft, Cambridge, UK) and had CCTAGGCTTCTCAGTGTC-30. Primer pairs used for Rps9 were 50-TCT r-values 40.95. P-values resulting from t-test comparisons of Ara-C- ATTCACCATGCCCGTGT-30 and 50-GGCGAACAATGAAGGATGG-30. resistant cells to their parental lines were o0.001.

Leukemia Letters to the Editor 1515 Table 2 Tenfold gene expression changes comparing Ara-C-resistant B117H cells to B117P parental cells

Probe ID Gene ID Description Fold change

B117H vs B117P

1439012_a_at Dck Deoxycytidine kinase À273.157 1426438_at Ddx3y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked À112.117 1449176_a_at Dck Deoxycytidine kinase À92.503 1452077_at Ddx3y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked À30.279 1426598_at Uty Ubiquitously transcribed tetratricopeptide repeat gene, Y À15.103 1419905_s_at Hpgd Hydroxyprostaglandin dehydrogenase 15 (NAD) À13.998 1421408_at Igsf6 Immunoglobulin superfamily, member 6 À13.870 1421571_a_at LOC100045833, Ly6c1, Ly6c2 Lymphocyte antigen 6 complex, locus C À13.366 1433930_at Hpse Heparanase À11.545 1426439_at Ddx3y DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked À11.326 1433944_at Hectd2 HECT domain containing 2 10.212 1421681_at Nrg4 neuregulin 4 10.217 1459860_x_at Trim2 Tripartite motif-containing 2 12.334 1446594_at Gm4371 Eukaryotic translation initiation factor 3, subunit I pseudogene 13.897 1439036_a_at Atp1b1 ATPase, Na+/K+ transporting, b1 polypeptide 13.990 1420822_s_at Sgpp1 Sphingosine-1-phosphate phosphatase 1 15.374 1420821_at Sgpp1 Sphingosine-1-phosphate phosphatase 1 17.392 1424292_at Depdc1a DEP domain containing 1a 31.617 1426236_a_at Glul RIKEN cDNA 5830403L16 gene 43.289

Table 3 Tenfold gene expression changes comparing Ara-C-resistant B140H cells to B140P parental cells

Probe ID Gene ID Description Fold change

B140H vs B140P

1456642_x_at S100a10 S100 calcium binding protein A10 (calpactin) À95.306 1416762_at S100a10 S100 calcium binding protein A10 (calpactin) À44.896 1438968_x_at Spint2 Serine protease inhibitor, Kunitz type 2 À36.941 1426980_s_at E130012A19Rik RIKEN cDNA E130012A19 gene À13.315 1422776_at Serpinb8 Serine (or cysteine) proteinase inhibitor, clade B, member 8 À11.758 1455865_at Insm1 Insulinoma-associated 1 À10.926 1434353_at Sfmbt2 Scm-like with four MBT domains 2 À10.866 1426523_a_at Gnpda2 Glucosamine-6-phosphate deaminase 2 À10.369

Conflict of interest 2 Shi JY, Shi ZZ, Zhang SJ, Zhu YM, Gu BW, Li G et al. Association between single polymorphisms in deoxycytidine kinase The authors declare no conflict of interest. and treatment response among acute myeloid leukaemia patients. Pharmacogenetics 2004; 14: 759–768. 3 Veuger MJ, Honders MW, Landegent JE, Willemze R, Barge RM. Acknowledgements High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia. Blood We acknowledge support from the Leukemia and Lymphoma 2000; 96: 1517–1524. Society of America (LLS 7019-04, Specialized Center of Research) 4 Song JH, Kim SH, Kweon SH, Lee TH, Kim HJ, Kim HJ et al. Defective expression of deoxycytidine kinase in cytarabine- and the Leukemia Research Fund (University of Minnesota). We resistant acute myeloid leukemia cells. Int J Oncol 2009; 34: especially thank Teng Zhang for his assistance with the drug assay 1165–1171. and Natashay J Bailey for her work on the growth assay. 5 Jiang Y, Prunier C, Howe PH. The inhibitory effects of disabled-2 (Dab2) on Wnt signaling are mediated through Axin. Oncogene SK Rathe and DA Largaespada 2008; 27: 1865–1875. Department of Genetics, Cell Biology, and Development, 6 Akyerli CB, Beksac M, Holko M, Frevel M, Dalva K, Ozbek U et al. The Masonic Center at the University of Minnesota, Expression of IFITM1 in chronic myeloid leukemia patients. Leuk Minneapolis, MN, USA Res 2005; 29: 283–286. E-mail: [email protected] 7 Kongkham PN, Northcott PA, Ra YS, Nakahara Y, Mainprize TG, Croul SE et al. An epigenetic genome-wide screen identifies SPINT2 as a novel tumor suppressor gene in pediatric medulloblastoma. References Cancer Res 2008; 68: 9945–9953. 8 Chuthapisith S, Bean BE, Cowley G, Eremin JM, Samphao S, 1 Yin B, Kogan SC, Dickins RA, Lowe SW, Largaespada DA. Trp53 Layfield R et al. Annexins in human breast cancer: possible loss during in vitro selection contributes to acquired Ara-C resistance predictors of pathological response to neoadjuvant chemotherapy. in acute myeloid leukemia. Exp Hematol 2006; 34: 631–641. Eur J Cancer 2009; 45: 1274–1281.

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