Expression of the leukemic prognostic marker CD7 is linked to epigenetic modifications in chronic myeloid leukemia
© Rogers et al; licensee BioMed Central Ltd. 2010
Received: 5 October 2009
Accepted: 22 February 2010
Published: 22 February 2010
Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells.
To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin-CD34+) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin-CD34+ cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin-CD34+ cells from CML patients with high CD7 expression.
These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.
A feature of chronic myeloid leukaemia (CML) is the Philadelphia chromosome (Ph). This abnormal chromosome results from a reciprocal translocation between chromosome 9 and 22 that gives rise to the BCR-ABL fusion gene that is now the accepted defining hallmark of the disease [1–3]. CML is typically diagnosed in an initial chronic phase (CP) which is characterized by the formation of a multi-lineage clone of Ph+/BCR-ABL+ leukemic cells that typically dominates the entire hematopoietic system by the time of diagnosis. This clone includes a selectively enlarged compartment of myeloid progenitors that produce an elevated number of normally differentiated granulocytes . Targeted therapy of CML with imatinib mesylate (IM) or other inhibitors of the BCR-ABL oncoprotein is the current therapy of choice for patients with CP disease, although IM does not always produce durable remissions [5, 6]. The most common cause of a poor response outcome is the appearance of IM-resistant cells. This resistance might be BCR-ABL-dependent, such as mutations in the kinase domain of BCR-ABLand genomic amplification of the BCR-ABL locus or BCR-ABL-independent mechanisms such as constitutive activation of downstream pathways [7–9]. Other genetic causes of disease heterogeneity as well as the inherent resistance of the leukemic progenitor/stem cells to IM may be additional contributing factors [2, 10, 11]. Identification and characterization of epigenetic changes that control the properties of CML cells, especially those of CML stem/progenitor (lin-CD34+) cells may be useful for the design of suitable therapies for IM-refractory CML .
As with other cancers, various markers divide CP CML into different subclasses that are associated with different patient survival probabilities. CD7 and elastase 2 (ELA2) are two such genes that show differential expression patterns in CML cells . Various studies indicate expression of CD7 to be upregulated in CML cells (and various other leukaemias and lymphomas) where it has been associated with poor survival [14, 15]. ELA2 is clustered with two other serine protease gene family members, azurocidin 1 and proteinase 3 (PRTN3, also known as Myeloblastin) genes, at chromosome 19pter . These three genes are expressed co-ordinately and their products packaged together into azurophil granules during neutrophil differentiation. High expression of these three genes in CML is associated with a favourable prognosis .
CD7 is located on chromosome 17 and encodes a cell surface glycoprotein member of the immunoglobulin superfamily. The protein is found on thymocytes and mature NK and T-cells [17, 18]. It plays an essential role in T-cell interactions and also in T-cell/B-cell interactions during early lymphopoeisis. ELA2 is one of six structurally similar human elastase genes, ELA1, 2, 2A, 2B, 3A and 3B. Elastases form a subfamily of serine proteases that hydrolyze many proteins in addition to elastin. ELA2 hydrolyzes proteins within specialized neutrophil lysosomes, called azurophil granules, as well as proteins of the extracellular matrix following the protein's release from activated neutrophils. In addition, ELA2 may play a role in degenerative and inflammatory diseases by its proteolysis of collagen-IV and elastin of the extracellular matrix. This protease degrades the outer membrane protein A (OmpA) of E. coli as well as the virulence factors of such bacteria as Shigella, Salmonella and Yersinia . Mutations in the ELA2 gene are associated with cyclic neutropenia and severe congenital neutropenia (SCN) .
The role of epigenetic mechanisms in the transcriptional control of CD7 and ELA2 is unknown. In this study we have examined the transcription pattern of these two genes in normal adult human bone marrow and primary CML cells. We specifically compared the degree of 5' region DNA methylation with CD7 and ELA2 transcript levels and assessed levels of histone acetylation at the promoter regions of CD7 and ELA2 in expressing and non-expressing cell lines.
Materials and methods
Clinical data from 12 CML patients studied
Age at diagnosis
WBC at diagnosis
Disease status at diagnosis
Light density cells were first isolated by centrifugation on ficoll-hypaque and then cells expressing the following lineage (lin) markers: CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A were removed immunomagnetically using a column (StemSep, StemCell Technologies, Vancouver, BC) as recommended by the manufacturer. Cells were then cryopreserved in DMSO plus FCS until required. After thawing, the lin- cells were stained with allophycoerythrin (APC)-conjugated anti-human CD34 antibody (8G12, Becton Dickinson, San Jose, CA,) and propidium iodide (PI, Sigma Chemicals, St. Louis, MO). Viable (PI-) lin-CD34+ cells (enriched in stem/progenitor cells) were then isolated using a FACSVantage flowcytometer (BD). Cells were further sorted, where indicated in the results, using fluorescein isothiocyanate (FITC)-conjugated anti-human CD7 antibody (M-T701, BD).
DNA and cDNA preparation
DNA was prepared in the following manner. Cell lines were washed twice in PBS, then boiled in sterile water for 10 mins. Samples were incubated with proteinase K for two hours, followed by denaturation for 10 mins. DNA was isolated from CML and normal tissue samples using the Qiagen (Germantown, MD) DNA/RNA isolation kit, according to the manufacturer's recommendation. The PicoPure™ RNA extraction kit (Arturus, Mountainview, CA) was use to extract RNA, according to the manufacturer's protocol using DNaseI (Invitrogen, Carlsbad, CA) treatment during the procedure to minimize contamination with genomic DNA. RNA was reverse transcribed with SuperScriptIII (Invitrogen) to generate first strand cDNA,
Bisulfite conversion and PCR amplification
Bisulfite conversion of DNA was performed using the EZ DNA methylation kit (Zymo Research, Orange, CA), according to the manufacturer's protocol, with minor modifications . Treated DNA was cleaned and eluted to 15 μl following the manufacturer's instructions. PCR amplifications of two regions spanning the start of the ELA2 gene were performed on converted DNA using the following primers: ELA2BS1 for 5'-TGGGTTTTATTTGGAAGAGATTTAG-3' with ELA2BS1rev 5'-CCTCCAAACAAAATTCAAAATACAC-3', and ELA2BS2for 5'-GTGTATTTTGAATTTTGTTTGGAGG-3' with ELA2BS2rev 5'-CCTCAATCTCTTCTAATCTCC-3' with Platinum Taq (Invitrogen Life Technologies) under the following conditions: 94°C for 8 minutes, followed by 40-45 cycles of 94°C for 90 seconds, 55°C for 90 seconds, and 72°C for 45 seconds.
PCR amplification of the region spanning the start of the CD7 gene was performed on converted DNA using the following primers: CD7BS for 5'-TAGAGGATTAGGTAGGTTG-3' with CD7BSrev 5'-AACTCTTACCTTAAACAACC-3', with the same conditions as above apart from a 50°C annealing step. A final elongation step of 10 minutes was included in all reactions. PCR products were analyzed by gel electrophoresis and products purified with MiniElute (Qiagen).
PCR products were cloned using the pGEM-T Easy kit (Promega, Madison, WI). Sequencing was performed by The McGill University and Genome Québec Innovation Centre Sequencing Platform. Only unique sequences (as determined by either unique CpG methylation pattern or unique non-conversion of non-CpG cytosines) are shown, and all sequences had a conversion rate of higher than 95%.
Combined bisulfite and restriction analysis (CoBRA)
In addition to cloning and sequencing, PCR products were digested individually with the restriction enzymes Hin fI, Mbo I, Taq I, or Rsa I, which contain CpG dinucleotides in their recognition sites. If the CpG is methylated in the original sample, then the restriction enzyme site will be conserved during bisulfite treatment and the PCR product will be digested. All enzymes were purchased from New England Biolabs (Ipswich, MA), and digests were performed overnight following manufacturer's instructions. Control reactions were performed each time to ensure complete digestion, and uncut reactions were run on agarose gels with digests for comparison.
Chromatin immunoprecipitation and quantitative PCR
Absolute expression analysis using quantitative realtime PCR
Absolute quantification of CD7, ELA2 and PRTN3 cDNAs was performed according to Jiang et al. (2004) . The following primers: ELA2 forward primer 5'-ACGACATCGTGATTCTCCAGCTCA-3', ELA2 reverse primer 5'-CGTTGAGCTCCTGCAGGAC-3', CD7 forward primer 5'-TAGACCCAGAGAGGCTCAG-3', CD7 reverse primer 5'-GGAGACTGCTGCACCTCTTGG-3'. The housekeeping gene GAPDH was amplified as an internal control using forward primer 5'-CCCATCACCATCTTCCAGGAG-3' and reverse primer 5'-CTTCTCCATGGTGGTGAAGACG-3'. 40 rounds of amplification with SYBR® Green PCR Master Mix (Applied Biosystems) were performed. The default 7500 System SDS software version 1.2.10 (7500 RealTime PCR System, Applied Biosystems) cycle was used with amplification performed at 62°C in a total volume of 25 μl. Dissociation curve analysis was performed after the end of each PCR to confirm the presence of a single and specific product.
P-values for the DNA methylation analysis were calculated using Fisher's exact test. The statistical significance of absolute quantities of ELA2 and CD7 cDNAs in normal and CML samples was calculated using the Mann-Whitney U-test.
Transcription of CD7 and ELA2 is associated with DNA hypomethylation in leukemic cell lines
We first screened a panel of human leukemic cell lines for transcription of CD7 and ELA2 with RT-PCR (Additional File 1). THP-1 transcribes ELA2 but not CD7, ALL-SIL transcribes CD7 and RPM1 is negative for ELA2. We then assessed whether methylation patterns of the 5' CpG-rich regions of the CD7 and ELA2 genes are linked with their transcriptional activity. We identified a CpG rich region in the human ELA2 gene, from 606 nt upstream of the transcriptional start site to 247 nt downstream, including all of exon 1. This region does not contain any CpG islands according to the standard definition (> 50% G+C content, and observed/expected CpG ratio of > 0.6). However, it is CpG rich, with an observed to expected ratio of 0.5, compared to 0.1 across the human genome. It is also GC rich (58%), not only compared to the genome average of 41% GC, but also compared to the 48% GC average across chromosome 19 .
DNA was isolated from all three of these cell lines and treated with sodium bisulfite. Sodium bisulfite converts cytosine to uracil, but does not convert 5-methylcytosine, allowing the bases to be distinguished by PCR amplification. The 853-bp CpG-rich region of the ELA2 gene was PCR amplified from the treated DNA, cloned and sequenced (Figure 1A). CoBRA analysis was also performed on the PCR products, using two enzymes, as shown in Figure 1A, allowing analysis of three of the CpG dinucleotides. If the CpG was originally methylated, the restriction enzyme site will be retained in the treated DNA and the PCR product will be digested. The results of both the sequencing and the CoBRA show that the CpG rich region is significantly less methylated in the ELA2+ THP-1 cell line than in the ELA2- RPM1cell line, in which the majority of the CpGs are methylated. We also analyzed the DNA methylation status of ELA2 in the non-expressing (Additional File 1A) CML cell line, K562 (Additional File 2A), showing moderate to high methylation.
We also identified a CpG-rich region in the human CD7 gene, from 277 nt upstream of the transcriptional start site to 200 nt downstream of the transcriptional start site, and performed a similar analysis on this region (Figure 1B). This region also does not contain any CpG islands, but it is enriched for CpGs, with an observed to expected ratio of 0.3. It is also GC rich (69%) compared to the 45.5% GC average across chromosome 17 . Bisulfite sequencing and CoBRA revealed that this region is significantly less methylated in the CD7+ ALL-SIL cell line than in the CD7- THP-1 cell line, in which the majority of CpGs are methylated. We also analyzed the DNA methylation status of CD7 in the low-expressing (Additional File 1B) CML cell line, K562 (Additional File 2B), which showed partial methylation.
Expression of CD7 and ELA2 is associated with histone acetylation in leukemic cell lines
Range of ELA2 and CD7 transcript levels in CD34+ cells from CML patients and normal controls
DNA hypomethylation correlates with increased CD7 expression on individual cells in CML
DNA methylation and transcript levels of ELA2 in CML are not correlated
In most cancers, epigenetic changes, as well as genetic aberrations, contribute to an altered program of gene expression, disease progression and increased tumor heterogeneity [26–28]. Genome-wide DNA hypomethylation leads to ectopic expression of oncogenes [29, 30] and a marked increase in genomic instability . Specific hypermethylation of tumor suppressor genes can act as the second hit or as the primary mechanism of transcriptional shutdown [32, 33]. Furthermore, transcription promoting and inhibiting modifications to histones also occur in many cancers. These widespread epigenetic changes justify the use of chromatin remodelling drugs such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors for the reactivation of epigenetically-silenced tumor suppressor genes. As a specific example, in CML and some Ph+ acute lymphoblastic leukemias, microRNA-203 (miR-203) is silenced by DNA hypermethylation . This miRNA acts as a tumor suppressor by down-regulating ABL through binding the 3' untranslated region (3'UTR) of ABL mRNA thereby preventing its translation .
In this study we assessed the epigenetic state of two genes, CD7 and ELA2 in primitive leukemic cells isolated from CP CML patients. Both of these genes were known to show altered expression in CML cells, and we confirmed this finding for primitive lin-CD34+ CML cells. The extent of altered expression of both genes has also been implicated as having prognostic significance. In three human leukemic cell lines, the extent of DNA methylation and histone acetylation of the CD7 and ELA2 promoters correlated with expression. However, we found a similar correlation to be limited to the CD7 gene in primitive CML cells. It is possible that the higher level of ELA2 transcript detected in some of the CML samples is contributed by a small subset of the CD34+ cells in which case an associated hypomethylation of the ELA2 gene would have been masked by the situation in the bulk of the cells. In addition, the difference in ELA2 transcript levels between the ELA2-high and ELA2-low expressing lin-CD34+ cells was relatively small as compared to the much larger differences (20,000-fold) exhibited between ELA2+ and ELA2- cell lines. Thus differences in DNA methylation state amongst the primary cells might not be detectable.
PRTN3 (also known as PR3) is located ~4 kb upstream of ELA2 on human chromosome 19 and its expression has been shown to be co-regulated with ELA2. Our results confirm this co-regulation as well as indicating that, at least in our patient cohort, the transcriptional regulation of these two genes is not uncoupled in CP CML patients (Figure 3B and 3C). There is some correlation between DNA methylation of the upstream and intragenic regions of PRTN3 and its expression during granulopoeisis . However, there is little evidence for a correlation between the amount of DNA methylation and relative abundance of PRTN3 transcripts in terminally differentiated granulocytes . Therefore, it is plausible that differential DNA methylation, at least at the promoter region, is not the primary means of transcriptional control and transcript quantity modulation for PRTN3 and ELA2. Our results highlight the importance of comparing data from cell lines with primary patient samples for a more physiologically relevant interpretation.
A region located in intron 2 of the PRTN3 gene was shown to have enhancer activity for the ELA2 gene . This enhancer element is located within a CpG island (UCSC genome browser) that stretches from exon 2 to intron 3 of PRTN3 and might have differential DNA methylation during granulopoeisis as well as in CML. There are two internal (intragenic) CpG islands within the ELA2 gene. The most 5' CpG island stretches from exon 2 to exon 3 of ELA2 gene (UCSC genome browser). Interestingly, a recent report covering all CpG islands of human genome shows differential methylation of this region in the primary tissue tested with the CpG island showing hypomethylation in one of two peripheral blood lymphocyte samples . This region represents a potential differentially methylated region with possible regulatory roles in the transcription of ELA2.
Our results show that the level of CD7 transcript is higher in CML lin-CD34+ cells than in their normal counterparts (Figure 3A). This finding is in accord with previous studies showing a higher proportion of CD34+ cells co-expressing CD7 in CML patients versus normal individuals [38–40]. It has also been shown that chromosomal anomalies in addition to the Ph chromosome are more commonly detected in the CD34+CD7+ subset of CML cells than in the CD34+CD7- Ph+ cells . We also show that the expression of CD7 is linked to the DNA methylation of its promoter region in both primary samples and cell lines. The hypomethylation of the CD7 promoter region in CD34+CD7+ cells compared to CD34+CD7- cells of the CML-1 and CML-10 patient samples (Figure 5) denotes a likely physiological role for DNA methylation in the transcriptional repression of this gene. Whether the expression of CD7 is due to specific demethylation of its promoter or just a by-product of the genome-wide DNA hypo-methylation is unknown. However, it seems that, at least in a portion of the CD7+ cells, only one allele is hypomethylated.
In this study, we have shown a link between epigenetic modifications and CD7 expression in CML. There is great variability in the survival outcome of CP CML patients despite no additional detectable chromosomal abnormalities beyond the BCR-ABL translocation [13, 41]. Changes in the epigenetic maintenance of genes such as CD7 could be an earlier indicator of disease progression. DNA methylation analysis can be performed reliably on as little as a few hundred cells, allowing analysis of rare cell populations. Since CD7 is an early prognostic marker detected at the CP stage, changes in its DNA methylation level, in conjunction to transcript levels, offer a potentially useful predictor of early-stage poor-prognosis CML. In light of the revived use of chromatin remodeling drugs in clinical studies, especially the various generations of DNA methyltransferase inhibitors, we believe that the state of DNA methylation of prognostic genes should be scrutinized more closely.
We sincerely thank Karen Lambie, Gayle Thornbury, Lindsay Laycock, Wenbo Xu, Liane Gagnier and Dianne Bamford for technical assistance and Dr. Afshin Raouf for sharing expertise. We also thank Drs. Donna Forrest and Clay Smith of the Leukemia BMT Service of the BC Cancer Agency and Dr. Mike Caligiuri of the Ohio State University Medical School, Ohio, USA for help in obtaining suitable samples. Core support was provided by the British Columbia Cancer Agency.
This work was supported by a grant from the Cancer Research Society to D.L.M.
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