Identification of Genes Potentially Involved in Disease Transformation

JJWM Janssen1, SM Klaver1, Q Waisﬁsz1, G Pasterkamp2, DPV de Kleijn2, GJ Schuurhuis1 and GJ Ossenkoppele1

1Department of Hematology, VU University Medical Center, Amsterdam, The Netherlands; and 2Department of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands

In patients with chronic myeloid leukemia (CML) who do not cases of advanced stage CML, N- and K-RAS mutations have reach a (near) complete cytogenetic response, the disease been reported.8 The question which of these changes occurred progresses over several years from an indolent, chronic phase into a rapidly fatal blast crisis. Events that are responsible for as a cause or as a result of the transformation itself has not been this transformation process are largely unknown. To identify solved yet. changes in gene expression that occurred during the course of To identify events responsible for disease progression, we the disease, we performed cDNA subtraction on sequentially performed PCR subtraction on samples from a CML patient of stored peripheral blood mononuclear cell pellets, collected whom peripheral blood mononuclear cells had been collected throughout the course of disease of a single CML patient. In throughout the course of the disease. A total of 32 differentially total, 32 differentially expressed sequences were identified, of which 27 corresponded to known genes. On quantitative PCR, expressed sequences were identified, of which eight potentially eight of these genes, YWHAZ, GAS2, IL8, IL6, PBEF1, CCL4, relevant genes showed similar differential expression in an SAT and MMRN, showed comparable differential expression in additional set of CML patients. additional CML patient samples. This set of genes can be considered as a starting point for further research on causes of disease transformation in CML and may lead to new targets in Materials and methods the treatment of resistant CML. Leukemia (2005) 19, 998–1004. doi:10.1038/sj.leu.2403735 Patients Published online 7 April 2005 Keywords: CML; transformation; subtractive hybridisation Suppression subtractive hybridization was performed on peripheral blood mononuclear cell pellets isolated at diagnosis, 2 years after diagnosis and at blast crisis of a single CML patient. This was a man of 51 years who was diagnosed with CML in Introduction 1995. Sokal score at diagnosis was 1.22, Euro score 2065. Cytogenetics revealed the standard translocation Chronic myeloid leukemia (CML) is a malignant disorder of the t(9;22)(q34;q11), RT-PCR showed a b3a2 fusion. He had no hemopoietic stem cell that is characterized cytogenetically by HLA-identical siblings and was given intensive treatment with the presence of the Philadelphia chromosome, which represents induction chemotherapy as part of a clinical research protocol 9 6 fusion of the BCR-gene derived from chromosome 22 with the in August 1995. Next, interferon alpha 3–6 Â 10 U s.c. q.d. ABL-gene from chromosome 9. The protein product of this was started, but because of lack of any response and intolerance fusion, BCR-ABL, has leukemogenic properties and is pivotal in to this drug, he switched to hydroxyurea after 1.5 years. At 3.6 the development of CML. Typically, after several years, the years after diagnosis, the disease transformed into a blast crisis chronic phase of the disease transforms into a rapidly fatal blast and he died of progressive bone marrow failure shortly crisis. In patients that obtain a (near-) complete cytogenetic thereafter. Blast cells at blast crisis were Sudan black positive response on interferon and/or imatinib, development of blast and were positive on immunophenotyping for CD34, CD13, crisis is delayed and may even be prevented. Although the CD117 and CD45 (dim), but negative for CD41, CD5, CD2, response percentage to imatinib is high, especially in patients CD19 and CD20, compatible with myeloid blast crisis. with high Sokal or Euro-risk scores, primary and secondary Characteristics of the index patient at the time points when resistance to the drug is seen. Moreover, a small number of samples for suppression subtractive hybridization were taken patients present with disease in accelerated or even blastic are shown in Table 1. Peripheral blood mononuclear cell pellets phase, in which imatinib is clearly less effective.1 of an additional group of CML patients were used to validate the The molecular mechanisms that underlie BCR-ABL-mediated differential expression of genes as found in the index patient. transformation are poorly understood; however, several events None of these patients ever reached any cytogenetic response. have been described to coincide with development of blast Treatment consisted of interferon-alpha and/or hydroxyurea. crisis. Mutations or deletions of P53 and its related gene TP73L Samples were collected throughout the course of the disease. (in around 25% of myeloid blast crises), doubling of the Late chronic phase was defined as a time point at least 2 years Philadelphia-chromosome, trisomy 8, upregulation of C-MYC after diagnosis and not fulfilling common criteria of blast crisis. and RB expression have been found.2–6 Changes in proliferation, Of several patients, sequential samples from diagnosis through- differentiation, apoptosis sensitivity, DNA repair function, as out blast crisis were available; in others, a more limited part of well as loss of imprinting and genomic instability have also been the disease course was represented, for example, from diagnosis observed upon disease transformation.7 In a small minority of to late chronic phase, or from late chronic phase to blast crisis.

Correspondence: Dr JJWM Janssen, Internist-hematologist, Depart- Isolation of peripheral blood mononuclear cells ment of Hematology, VU University medical center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands; Fax: þ 31 20 444 2601; E-mail: [email protected] Peripheral blood was collected by venipuncture into lithium Received 18 August 2004; accepted 31 January 2005; Published heparin-coated glass containers. Peripheral blood mononuclear online 7 April 2005 cells were isolated using density gradient centrifugation with Genes involved in disease transformation of CML JJWM Janssen et al 999 Table 1 Clinical characteristics of index patient and directly sequenced thereafter on the ABI Prism 310 Genetic Analyzer (Applied Biosystems-Perkin Elmer, Foster Year City, CA, USA).

1995 1997 1999 Quantitative PCR (Q-PCR) Diagnosis Late chronic phase Blast crisis

Total leukocytes ( Â 109/l) 176 7.5 57.9 PCR amplification was performed with a LightCycler real-time Thrombocytes ( Â 109/l) 368 275 2 PCR machine (Roche Diagnostics, Almere, The Netherlands). Basophils (%) 1 6 9 Reaction volumes were 20 ml, consisting of 2 ml cDNA, 2 mlof Blasts (%) 2 2 85 LightCycler Fast Start DNA SYBR Green Mastermix (Roche) and Promyelocytes (%) 5 2 0 0.5 mM reverse and forward primers each. MgCl2 was added to a Myelocytes (%) 22 8 0 m Metamyelocytes (%) 10 6 1 final concentration of 4 M in all reactions except for GAPDH Stabs (%) 9 10 0 amplification to which 1.5 mM was added. In all PCR reactions, Segments (%) 43 46 1 conditions were an initial denaturation step at 951C for 10 min, Monocytes (%) 2 14 2 followed by 45 cycles, each for 15 s at 951C, 10 s at 591C and Ph+ (%) 100 100 ND 10 s at 721C. For quantification, comparisons were made with ND: not done. standard curves of PCR products cloned into PGEM-T-easy vectors (Promega). GAPDH signals were used for normalization of expression for highly expressed genes, Beta-glucuronidase Ficoll-Paque (1.077 g/ml). Cells were pelleted, snap-frozen in (GUS) signals for normalization of genes with a relative low liquid nitrogen and then stored at À801C until use. expression. For primer design the GeneFisher program, acces- sible at http://bibiserv.techfak.uni-bielefeld.de/genefisher/ was used.11 Primers were purchased from Isogen (Maarssen, The Suppression subtractive hybridization and library Netherlands). Primer sequences are shown in Table 2b. screening Statistical analysis Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Westburg BV, The Netherlands) according to the manufacturer’s Gene expression levels as expressed relative to GUS or GAPDH protocol. cDNA synthesis, subtraction PCR, library screening copy numbers were log -transformed. Median values of the and sequence analysis were performed as described pre- e additional groups of patients at the three different disease stages viously.10 Briefly, cDNA was prepared by using the SMART were compared using the Mann–Whitney U-test for nonpara- PCR cDNA synthesis kit (Clontech, Beckton Dickinson Bios- metric variables. A P-value of less than 0.05 was considered to ciences BV, Woerden, The Netherlands) and samples were indicate a statistically significant difference. mixed for the PCR-Select cDNA subtraction kit according to the manufacturer’s protocol. Subtracted cDNA products were subsequently ligated into pGEM-T-Easy cloning vectors (Prome- Results and discussion ga Benelux BV Leiden, The Netherlands) and transformed into supercompetent XL1Blue cells (Stratagene Europe, Amsterdam, Differential expression of the identified genes was validated in The Netherlands). Plasmid DNA was spotted in an identical the index patient samples by Q-PCR. The results from manner onto four separate nylon membranes (Hybond-N, subtractive hybridization and Q-PCR correlated for all tested Amersham Pharmacia) for every subtraction procedure. This genes, except for MEN1, which showed only weak differences was performed with libraries derived from the subtraction of on the library screening blots. Table 3 gives the extent of (I) chronic phase minus diagnosis and reverse, (II) late chronic differential expression for all tested genes. Four genes showed phase minus blast crisis and reverse and (III) blast crisis minus less than two-fold difference, which was considered too low for late chronic phase and reverse. After baking the membranes at reliable determination of differential expression. The differences 801C for 2 h, each blot was hybridized with a different radioactively labeled cDNA probe: for the subtraction procedure of chronic phase minus diagnosis, blot 1 was hybridized Table 2a Primer sequences p53 with unsubtracted cDNA of the late chronic phase sample, blot 2 with unsubtracted cDNA of the diagnosis sample, blot 3 with Exon 5 forward 50-GGAATTCTCTGTTCACTTGTGCCCTGACTTT-30 0 0 subtracted DNA of chronic phase minus diagnosis and blot 4 Exon 5 reverse 5 -GGAATTCGGGCAACCAGCCCTGTCGTC-3 Exon 6 forward 50-GGTTGCCCAGGGTCCCCAG-30 with subtracted cDNA of diagnosis minus chronic phase. Exon 6 reverse 50-CGGAGGGCCACTGACAACCA-30 Likewise, hybridizations were performed for all subtraction Exon 7 forward 50-CTTGCCACAGGTCTCCCCAA-30 procedures. Only clones that showed a clear difference between Exon 7 reverse 50-AGGGGTCAGCGGCAAGCAGA-30 the two subtracted probes and no or a low signal for the Exon 8 forward 50-ACCTGATTTCCTTACTGCCTCTTGC-30 unsubtracted probes were selected for sequence analysis. Exon 8 reverse 50-GGCATAACTGCACCCTTGGTCTCCT-30 Exon 9 forward 50-AGGAGACCAAGGGTGCAGTTATGCC-30 Exon 9 reverse 50-ACCAGGAGCCATTGTCTTTGAGGC-30

P53 sequence analysis Sequence primers Exon 5 50-TCTCTCCAGCCCCAGCT-30 For P53 sequence analysis, DNA from mononuclear cell Exon 6 50-CAACCACCCTTAACCC-30 pellets was isolated with the QIAamp DNA Blood Mini Kit Exon 7 50-GCTGGGGCACAGCAGGC-30 0 0 (Qiagen) according to the manufacturer’s protocol. P53 exons Exon 8 5 -CTTCTTGTCCTGCTTGC-3 Exon 9 50-TTAGACTGGAAACTTTC-30 5–9 were ampliﬁed using PCR (primer sequences: see Table 2a)

Leukemia Genes involved in disease transformation of CML JJWM Janssen et al 1000 Table 2b Primer sequences Q-PCR Table 3 List of differentially expressed genes in index patient and level of differential expression APEX1 F 50-TGGCAAACCTGCCACACTCAAG-30 APEX1 R 50-CCAGGCAGCTCCTGAAGTTCAG-30 Name of gene Accession number Fold difference (by Q-PCR)a CCL4 F 50-CAGCGCTCTCAGCACCAATGG-30 CCL4 R 50-GATCAGCACAGACTTGCTTGCTTC-30 Diagnosis4blast crisis CD96 F 50-CCACTCCTCAACCCAGCAATTCC-30 PRG1 NM_002727 ND CD96 R 50-GGCTGTGCTGGTAAAGACATTGTC-30 IVNS1ABP NM_006469 o2(o0.7) CHI3L2 F 50-TCTTGACTGCGGGCGTATC-30 MPO NM_000250 ND CHI3L2 R 50-CCCAAGACCCATGGAAGTCA-30 DC2 F 50-CTGGTGGTGGTGTCTTACTTCC-30 Diagnosisoblast crisis DC2 R 50-GAAGCTGGATGCAAGTCCTTCC-30 EMR1 NM_001974 o2(o0.7) DNAJB1 F 50-TACACATTCCATGGAGACCCTCATG-30 CD96 NM_198196 3 (1.1) DNAJB1 R 50-GGCCAAAGTTCACGTTGGTGAAG-30 GAS2 NM_005256 12 (2.5) EMR1 F 50-GCATCTTTTTGGAAACCCTCAG-30 CHI3L2 NM_004000 11 (2.4) EMR1 R 50-AGTCCAACGTCACATTCTCTTC-30 FPR 1 F 50-CCTGCTGTATCTGCCTGGCTA-30 Diagnosis4late chronic phase FPR 1 R 50-GAATCCAGCCACCCAGATCA-30 DNAJB1 NM_006145 35 (3.6) GAPDH F 50-AGGTCATCCCTGAGCTGAACGG-30 HSPA8 NM_006597 5 (1.6) GAPDH R 50-CGCCTGCTTCACCACCTTCTTG-30 NXF1 NM_006362 2 (0.7) GAS2 F 50-CTGCCGAATGCTGCAGATC-30 DC2 NM_021227 o2(o0.7) GAS2 R 50-TGGCAGAGACCACCAAGTAG-30 APEX1 NM_080648 4 (1.4) GUS F 50-GAAAATATGTGGTTGGAGAGCTCATT-30 RPS6 NM_001010 o2(o0.7) GUS R 50-CCGAGTGAAGATCCCCTTTTTA-30 EEF1A1 NM_001402 3 (1.1) HSPA8 F 50-ATTGCAGAAGCCTACCTTGG-30 FLJ25286b NM_152546 ND HSPA8 R 50-CGTTTCTTTCTGCTCCAACC-30 IL6 F 50-CCAGAGCTGTGCAGATGAGT-30 Diagnosisolate chronic phase IL6 R 50-TCAGCAGGCTGGCATTTGT-30 SAT NM_002970 7 (1.9) IL8 F 50-GGCTCTCTTGGCAGCCTTCC-30 CCL4 NM_002984 106 (4.7) IL8 R 50-GCGCAGTGTGGTCCACTCTC-30 YWHAZ NM_145690 3 (1.1) MEN1 F 50-AGAAGGTCTCCGATGTCATATGG-30 PBEF1 NM_182790 7 (1.9) MEN1 R 50-ACCGGAGCTGTCCAATTTGG-30 IL6 NM_000600 42 (3.7) MMRN F 50-CAGATTTAAGGCGTTGGAAGC-30 IL8 NM_000584 9 (2.2) MMRN R 50-GTAGCATTCAGTTCTGACACAC-30 NACSINF 50-CCACCAGTCCTCTCACCAAA-30 Late chronic phase4blast crisis NACSINR 50-TTGGCTTTCGCCCCAAAAC-30 IL8 NM_000584 2 (0.7) NXF1 F 50-AGAGGCGGTTCTGGTATTCG-30 S100A8 NM_002964 ND NXF1 R 50-GCACAGTAACATGAATGCGATC-30 FPR1 NM_002029 ND IVNS1ABP F 50-ACCGTTGTAATGCAGGAGTGTG-30 LYZ NM_000239 ND IVNS1ABP R 50-TCACAGACTGCAGACTGGTGTC-30 MEN1 AF001893 c PBEF1 F 50-CAAGGACCCAGTTGCTGATC-30 PBEF1 R 50-CTCCTTTTCCTTCCTCCAGTG-30 Late chronic phaseoblast crisis SAT F 50-CACAGCATTGTTGGTTTTGC-30 MMRN NM_007351 6 (1.8) SAT R 50-GGATGGTTCATTCCATTCTGC-30 NACSIN NM_015252 24 (3.2) YWHAZ F 50-GAAAGCCTGCTCTCTTGCAAAGAC-30 LARS NM_020117 ND YWHAZ R 50-CCTCCTTCTCCTGCTTCAGCTTC-30 FLJ25286b NM_152546 ND C5orf13d NM_004772 ND C15orf15d NM_016304 ND FLJ10849b NM_018243 ND a Between brackets: loge-transformed difference. in expression between time points for the remaining genes b varied from 2- to 106-fold (linear values). Although possibly Genes hypothetically encoding proteins. cReverse to subtraction result. interesting, expressed sequence tags and genes that were only dOrf, open reading frame; ND, not done; NS, nonsignificant; predicted were not validated by quantitative PCR. D, diagnosis; LCP, late chronic phase; BC, blast crisis. Some of the identified differentially expressed genes pre- sumably represent differences in maturation stages between undifferentiated blasts at blast crisis and more mature myeloid time point of the disease for each gene. For 14 genes, progenitors at diagnosis and late chronic phase. Their identifica- subtraction results could not be confirmed in this additional tion demonstrates the power of the suppression subtractive group of patients. However, YWHAZ, GAS2, IL8, IL6, PBEF1, hybridization technique; however, these genes are probably CCL4, SAT and MMRN showed a similar pattern of expression in irrelevant to the disease transformation process. These genes the additional patient samples as was found in the index patient. include: MPO, LYZ, encoding the cytoplasmic enzymes Figure 1a–h shows the median relative expression levels of these myeloperoxidase and lysozyme, respectively; PRG1, encoding genes in this set of patient samples as observed in samples taken a proteoglycan that is stored in more mature myeloid cells;12 at diagnosis, late chronic phase or blast crisis. These consistently FPR1, a neutrophil G-protein-coupled receptor;13 S100A8, differentially expressed genes will be discussed in more detail encoding calgranulin A that is abundantly expressed in below. neutrophil cytoplasm.14 The upregulation of LARS (leucyl tRNA synthetase)15 was interpreted as reflecting increased transcript- ional activity in blast crisis cells. Apoptosis related genes The differential expression of the other genes was verified by Q-PCR in an additional group of CML patients. At least six Delayed apoptosis is a feature of early phase CML,16 but even (range 6–47) individual patient samples were analyzed for every more so of advanced disease stages in which upregulation of

Leukemia Genes involved in disease transformation of CML JJWM Janssen et al 1001 YWHAZ a e PBEF1 5.0 p = 0.045 p = 0.005 p = 0.01 2.0 p = 0.01 4.0 1.6 3.0 1.2 2.0 0.8 1.0 0.4 n=19 n=14NS n=11 n=9n=6 n=13 expression (% of GAPDH) expression expression (% of GAPDH) expression 0.0 0.0 D LCP BC D LCP BC

b GAS2 f 6.0 CCL4 p = 0.025 p = 0.006 p = 0.027 5.0 2.0 p = 0.008 1.6 4.0

1.2 3.0

0.8 2.0 n=36 n=26 0.4 n=46 1.0 n=11 n=9 n=10

expression (% of GUS) expression 0.0 0.0 D LCP BC D LCP BC c g SAT p = 0.044 IL8 p < 0.0001 p = 0.009 4.0 p < 0.001 NS 5.0

3.0 4.0

2.0 3.0

2.0 1.0 n=47 n=38 n=26 1.0 n=11 n=7 n=9

expression (% of GAPDH) expression 0.0 D LCP BC (% of GUS)expression 0.0 (% of GUS) expression D LCP BC d 4.0 IL6 h MMRN p = 0.021 p = 0.029 p = 0.02 3.0 5.0 p = 0.02 4.0 2.0 3.0 1.0 n=17 n=10 n=11 2.0

0.0 1.0 n=11 n=9 n=9 expression (% of GUS) expression D LCP BC expression (% of GUS) expression 0.0 D LCP BC -1.0

Figure 1 (a–h) Median relative expression levels of consistently differentially expressed genes as observed in samples taken at diagnosis, late chronic phase or blast crisis. Expression levels have been loge-transformed and are relative to the expression of GUS (Beta-glucuronidase) or GAPDH (Glyceraldehyde-3-phosphate dehydrogenase). Abbreviations: YWHAZ ¼ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase; GAS2 ¼ growth arrest speciﬁc-2; IL8 ¼ interleukin-8; IL6 ¼ interleukin-6; PBEF1 ¼ Pre-B-cell colony-enhancing factor precursor; CCL4 ¼ small inducible cytokine A4; SAT ¼ spermidine/spermine-N-acetyltransferase; MMRN ¼ multimerin. NS ¼ not signiﬁcant. D ¼ diagnosis; LCP ¼ late chronic phase; BC ¼ blast crisis.

BCR-ABL is a common event.17.YWHAZ (tyrosine 3-monooxy- chronic phase (Figure 1a). YWHAZ has a central role in the genase/tryptophan 5-monooxygenase;14-3-3 zeta protein) is in- regulation of apoptosis, cell growth and cycling by modulating volved in the regulation of apoptosis. Subtractive hybridization the functional activities of several key signaling proteins.18 For revealed higher expression in late chronic phase than at instance, the proapoptotic protein BAX can bind to the YWHAZ diagnosis. Q-PCR results in the group of CML patients at three protein, which leads to increased levels of homodimerized disease stages show that YWHAZ expression peaks at late BCL2 and hence to decreased apoptosis. A YWHAZ family

Leukemia Genes involved in disease transformation of CML JJWM Janssen et al 1002 GAS2 in blast crisis stitutively active IL6 induces continuous JAK/STAT activation in 2.8 AML.26 This, in turn, induces cell cycle transition through 2.4 upregulation of cyclins D2, D3, A and cdc25A, and the 27 2.0 concomitant downregulation of p21 and p27. It may be hypothesized that constant IL6 stimulation in chronic phase 1.6 CML induces increased proliferation, which could predispose to 1.2 secondary abnormalities that can culminate in blast crisis. 0.8 Otherwise, low IL6 levels at diagnosis and at blast crisis may be 25 GAS2 (% of GUS) 0.4 considered to reflect inadequate monocyte function. PBEF1 (Pre-B-cell colony-enhancing factor precursor) is, like 0.0 IL6 and IL8, highest expressed in late chronic phase (Figure 1e). MYELOID LYMPHOID/BIPHENOTYPIC Previous reports showed the gene to be expressed in bone Figure 2 Median expression levels of GAS2 in myeloid vs marrow stromal cells and in activated lymphocytes. PBEF1 lymphoid or biphenotypic blast crisis. Expression levels have been stimulates the growth of pre-B-cell colonies but no effect was loge-transformed and are relative to the expression of GUS. seen on myeloid or erythroid colonies.28 Using a comparable suppression subtractive hybridization as ours, it was found to be upregulated in colonic carcinoma samples as compared to member, BAP-1, shows 80% homology with YWHAZ and is normal mucosa.29 What role it may play in CML cells remains essential for the transforming properties of BCR-ABL.19 Although speculative, but if PBEF1 also increases proliferation of myeloid this association has not been described for the YWHAZ protein, progenitors, this might, as already stated for IL6, facilitate the it may similarly be involved in the BCR-ABL-induced apoptosis occurrence of secondary genetic events. resistance. Remarkably, also the CC chemokine gene CCL4 (‘small The protein product of the GAS2 (growth arrest specific-2) inducible cytokine A4’; SCYA4; MIP-1 beta) showed highest gene, whose expression has not previously been reported in expression at late chronic phase, while expression at both hematopoietic cells, is a caspase-3 substrate. It plays a role in diagnosis and blast crisis were clearly lower (see Figure 1f). the regulation of microfilament and cell shape changes during CCL4 is an antagonist of the proliferation-inhibiting effect of apoptosis.20 Overexpression of the protein stabilizes P53 and MIP-1 alpha30 and induces cytotoxic T- and NK-cell prolifera- thereby increases cell susceptibility to apoptosis following tion and cytolytic capacity.31 DNA-damaging agents.21 Therefore, the upregulation of GAS2 in blast crisis compared to diagnosis seems to be contradictory to its known physiological role. Q-PCR in a large group of Other genes patients shows a remarkable difference in expression between diagnosis and blast crisis, as was found by subtractive Higher expression of the growth- and cell cycle-associated hybridization, but even more between late chronic phase and gene SAT (spermidine/spermine N-acetyltransferase) in late blast crisis (see Figure 1b). Separating blast crises into myeloid chronic phase compared to diagnosis was found at subtractive vs lymphoid or biphenotypic reveals higher, although non- hybridization. Q-PCR in additional patient samples reveals significant GAS2 expression in myeloid crises (Figure 2). As P53 that expression at diagnosis is lowest compared to the mutations mainly occur in myeloid blast crisis, we hypothesized other two disease stages (see Figure 1g). SAT encodes a rate- that high GAS2 expression might represent P53 abnormalities. limiting enzyme in the catabolism of polyamines, substances Therefore, exons 5–9 of the P53 gene (the hot spot region involved in many cellular processes, including chromatin for mutations) were sequenced in the blast crisis sample of condensation, maintenance of DNA structure, RNA processing, the index patient, but no mutations were found (results translation and protein activation. Upregulation of SAT leads not shown). to apoptosis in melanoma cells.32 Its role in CML remains speculative. The gene for MMRN (multimerin), encoding a specific factor Cytokines V/Va-binding protein, was upregulated at blast crisis compared to late chronic phase according to the subtractive hybridization Some genes encoding proliferation-inducing cytokines and and this was confirmed by Q-PCR in the additional patient interleukins were also identified as being higher expressed in samples (Figure 1h). The expression levels at diagnosis were late chronic phase than at both diagnosis and blast crisis, such as comparable with those at late chronic phase. Multimerin is a IL8. Consistent results were found with Q-PCR in other CML constituent of platelet alpha granules33 and thus elevated patients (see Figure 1c). IL8 has mitogenic, motogenic and expression of this gene in blast cells may reflect differentiation angiogenic properties.22 A high level of expression has been into the megakaryocytic lineage.34 However, immunopheno- related to disease progression in CLL23 and it was strongly linked typing showed negativity for the megakaryocytic marker CD41 to refractory disease in T-ALL.24 If high expression in late on the blasts of all five patients where it was tested. chronic phase CML has relevance for CML transformation as Previous approaches to elucidate mechanisms of disease well is unknown but warrants further study. transformation with similar techniques as described by us found A second cytokine gene, IL6, was much higher expressed in downregulation of, among others, PIASy, a potential inhibitor of late chronic phase compared to both diagnosis and blast crisis in the STAT proteins with apoptosis-promoting and growth the index patient, as well as in the additional group of patients suppressive properties35 and upregulation of PRAME36 in blast (see Figure 1d). In agreement with this, it has previously been crisis progenitor cells derived from the bone marrow. In another, shown that IL6 levels were higher in cultures of CML monocytes probably less appropriate experiment, overexpression of ribo- of treated CML than those obtained at diagnosis and at blast somal proteins, of a c-myc-related protein and of a cellular crisis.25 At blast crisis, levels were very low, although the adhesion molecule was found in K562 cells, serving as a model difference with late chronic phase was not significant. Con- of CML blast crisis, compared to primary CML spleen cells. This

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