LEDGF/p75 has increased expression in blasts from chemotherapy-resistant human acute myelogenic leukemia patients and protects leukemia cells from apoptosis in vitro
© Huang et al; licensee BioMed Central Ltd. 2007
Received: 03 January 2007
Accepted: 23 April 2007
Published: 23 April 2007
Relapse due to chemoresistant residual disease is a major cause of death in acute myelogenous leukemia (AML). The present study was undertaken to elucidate the molecular mechanisms of chemoresistance by comparing differential gene expression in blasts from patients with resistant relapsing AML and chemosensitive AML.
About 20 genes were identified as preferentially expressed in blasts pooled from patients with resistant disease, as compared to chemosensitive AML blasts, based on differential gene expression screening. Half of these genes encoded proteins related to protein translation, of these a novel protein related to the ribosomal stalk protein P0. Other upregulated mRNAs coded for cytochrome C oxidase III, the transcription factors ERF-2/TIS11d, and the p75 and p52 splice variants of Lens Epithelial Derived Growth Factor (LEDGF). Analysis of blasts from single patients disclosed that LEDGF/p75 was the most consistently upregulated mRNA in resistant AML. Transfection experiments demonstrated that LEDGF/p75 and p52b antagonized daunorubicin-induced and cAMP-induced apoptosis in an AML cell line. Also HEK-293 cells were protected against daunorubicin by LEDGF/p75 and p52b, whereas LEDGF/p52 splice variants lacking exon 6 had proapoptotic effects. Interestingly, full length LEDGF/p75 protected against truncated pro-apoptotic LEDGF/p75.
Our results provide evidence for an association between the overexpression of genes encoding survival proteins like LEDGF/p75 and chemo-resistance in acute myelogenous leukemia. LEDGF/p75 has previously not been shown to protect against chemotherapy, and is a potential drug target in AML.
Acute myeloid leukemia (AML) is an aggressive malignant disorder characterized by neoplastic proliferation and accumulation of immature myeloid cells. Most patients achieve complete hematological remission after initial induction chemotherapy, but a large part of these patients will later develop leukemia relapse due to chemotherapy-resistant residual disease [1–3]. The overall long-term (5 years) AML-free survival is therefore less than 50% even for younger patients who receive the most intensive therapy [2, 3]. Chemoresistance can manifest either as primary resistance to the induction therapy or as AML relapse following initial chemotherapy-induced hematological remission. A high frequency of AML relapse is especially observed in certain patient subsets characterized by persistent leukemic disease after the initial induction cycle or by high-risk cytogenetic abnormality [4, 5]. Many of these abnormalities affect genes that encode proteins involved in the regulation of gene transcription . Chemoresistance after treatment with anticancer agents may also be linked to perturbed gene expression [6, 7]. These observations are consistent with altered gene expression being involved in chemotherapy resistance. This notion is further supported by two recent clinical studies describing association between long-term disease-free survival and particular gene expression profiles identified by cDNA microarray screening [8, 9]. The aim of the present study was to identify genes overexpressed in chemoresistant AML and in native AML cells derived from patients with documented or high risk of clinical chemoresistance. For this purpose, we used differential hybridization of pooled RNA transcripts and confirmed the findings for each individual patient using RNA dot-blot and RT-PCR analysis. Our study demonstrated increased expression in chemoresistant cells of several gene products, notably lens epithelial-derived growth factor (LEDGF)/p75 and a novel gene called A ML r esistance a ssociated p rotein (ARAP), similar to ribosomal protein P0. Enforced expression of the LEDGF/p75 and p52b genes protected against anthracycline-induced apoptosis. Interestingly, other less expressed splice variants of LEDGF were strong inducers of apoptosis, but their effect was overcome by co-expression of the full length variant. Our study suggests that AML cells express so far little known or unknown gene products able to protect against chemotherapy, and that naturally occurring pro-apoptotic splice variants can give clues to domains of these proteins that can be pharmacological therapy targets.
Identification of genes overexpressed in AML relapse cells
Genes preferentially expressed on chemotherapy-resistant AML relapse
Primary differential screening mRNA expression
Individual patients means of mRNA expression
We have recently found profound differences in phosphoprotein signaling cascades between AML cells from patients with low-risk and high-risk of relapse . Our L-cells were from low-risk patients, and their expression of 9 of the genes presented in Table 1 was studied in cells from individual patients. For comparison, we studied the expression of the same genes in cells from individual patients with high risk (H-cells) of relapse and the cells from patients with resistance (R-cells) to chemotherapy regimens (Table 1). It appeared that ARAP expression was enhanced in 4 of the 5 R-cell patients, average more than 4-fold, but not increased in the H-cells. High expression of this gene could therefore be a consequence of the chemotherapy. The genes encoding the transcription factors LEDGF/p75 and ERF-2 were also generally more upregulated in the R-cells than in the H-cells (Table 1). The other genes studied were upregulated in at least half of the patients whose R- and H-cells were studied. They were generally equally or more upregulated in the H-cells than in the R-cells, suggesting that previous chemotherapy was not required for the overexpression. We conclude that high-risk and low-risk AML cells differ not only with respect to phospho-signalomics , but also with respect to expression of the genes associated with AML relapse. Most R and H patients had increased expression of a number of the resistance-associated genes. We focused in more detail on the LEDGF/p75 because it had the highest relative expression in the R-cells relative to the L-cells among the differentially expressed genes tested (Table 1) and earlier studies indicating LEDGF/p75 to be involved in cell survival [14, 15].
LEDGF/p75/p52 mRNA splice variants in human AML cells
LEDGF/p75 can be expressed in a number of splice variants in AML cells. We wanted to know the possible functional significance of the overexpression of LEDGF/p75 in AML cells from patients with chemoresistance, and the significance of the splice variants of p52 described. For this purpose, we first used Flp-In 293 cells transfected with the various genes and analyzed for induction of spontaneous apoptosis or for ability to modulate the apoptosis induction by daunorubicin.
Overexpression of LEDGF/p75 protected against daunorubicin-induced apoptosis, whereas LEDGF/p52 splice variants without exon 6 were pro-apoptotic
Overexpression of LEDGF/p75 and p52b in IPC81 AML cells protected against apoptosis induced by daunorubicin or cAMP analog
Effects of daunorubicin on LEDGF/p75 expression in human NB4 AML cells
The present differential gene display analysis identified a number of genes with increased expression in relapsed AML. The increase of each mRNA varied between patients, in keeping with our recent study  which revealed different response of signaling pathways coupled to translational control in a similar panel of patient AML cells. Also array-based AML gene expression profiling studies [9, 22–24] underscore the heterogeneity of AML and the need for personalized therapy [25, 26].
A strikingly high proportion of the genes with increased expression in relapsed AML (Fig. 1) were related to protein translation. One of them, termed ARAP, has sequence similarities to ribosomal protein P0 and has not previously been cloned. The elucidation of ARAP function and the role proteins involved in protein translation will be the subject of future studies.
Importantly, we found increased mRNA levels in relapsed AML for the general transcriptional co-activator lens epithelium derived growth factor (LEDGF)/p75 and its splice product p52 . The LEDGF/p75 mRNA was the most consistently upregulated in our series of relapsed AML. AML cells transfected with LEDGF/p75 or p52b were significantly more resistant to apoptosis induction in vitro by either daunorubicin or the presumed physiological apoptogen cAMP (Fig. 5). Detailed studies in HEK293 cells showed that overexpression of LEDGF/p75 protected against daunorubicin-induced death to a similar extent as bcl-2 (Figs. 3, 4), suggesting that increased LEDGF/p75 might enhance chemotherapy resistance also in non-AML cells. This finding is consistent with LEDGF/p75 protecting cells against stress such as serum deprivation . LEDGF/p75 and p52 share the same 325 N-terminal residues, in which is found a PWWP domain (residues 1–100) interacting with stress response elements, a nuclear localization signal (residues 146 – 156), and an AT-hook motif (residues 179–198) . Although p52 only has 8 unique C-terminal amino acids, it can interact selectively with the splicing factor ASF/SF2 . The unique C-terminal 205 residues of LEDGF/p75 harbor a hepatoma derived growth factor homology domain that binds to chromatin even in the absence of a functional NLS . The pro-survival function of LEDGF/p75 is believed to depend on the 44 C-terminal residues since LEDGF/p75 (1–486) failed to protect hepatoma cells against serum deprivation . We found that p52b, which lacks the C-terminal residues of LEDGF/p75 (Fig. 2) did not induce apoptosis but our data suggest that it has some protective activity against daunorubicin-induced death (Fig. 4). This suggests that the C-terminal 205 residues of LEDGF/p75 were dispensable for protection against cell death. In fact, enforced expression of LEDGF/p75 (317–530) not only failed to protect against daunorubicin but induced significant death by itself (Fig. 4). The finding that p52 is not pro-apoptotic helps understand why patient leukemic cells with cytogenetic abnormality, t(9;11), involving the NUP98 gene and encoding NUP98-LEDGF/p75/p52 fusion proteins, have a high expression of both the LEDGF/p75 and p52 fusion products [28–30].
LEDGF/p75 and p52 were also present in NB4 AML cells, in which we detected four additional novel p52 variants, three of which had deleted exon 6. One variant (p52bΔE6) lacked only exon 6, while the others (p52Δ1, p52bΔ1) also lacked exon 7 and parts of exons 5 and 8 (Fig. 2). Exon 6 codes for residues important both for the NLS and the AT-hook of LEDGF/p75, and its absence will therefore presumably abolish or alter the nuclear localization of p52. All three p52 splice variants lacking exon 6 enhanced rather than protected against daunorubicin- induced death (Fig. 4). This means that the LEDGF/p75 gene can create both pro- and anti-apoptotic proteins. We found that full-length LEDGF/p75 protected against all three pro-apoptotic splice variants of p52 as well as against the pro-apoptotic p75 (317–530) fragment of LEDGF/p75 (Fig. 4). This suggests that increased expression of full-length LEDGF/p75 may protect against pro-apoptotic p52 splice variants as well as against truncated LEDGF/p75 variants produced by caspase-dependent processing of LEDGF/p75 . The high expression of LEDGF/p75 in relapsed AML may therefore protect against apoptosis both by stimulating the transcription of other survival genes and by partially blocking the effect of pro-apoptotic shorter versions of LEDGF/p75 and p52.
In summary, relapsed as well as "high risk" AML cells have increased expression of several genes encoding novel as well as known anti-apoptotic proteins. Few of these genes have previously been implicated in AML drug resistance. The LEDGF/p75 gene, as well as several pro-apoptotic splice variants of p52, in NB4 and AML (IPC81) cells have not been described before. A striking observation is the heterogeneity with regard to which genes that are upregulated, identifiable in the present study. This confirms that AML is a heterogeneous disease. We consider LEDGF/p75 to be a potential, novel therapeutic target in relapsed AML since it was among the genes most consistently upregulated and because it provided resistance towards daunorubicin after transfection. The LEDGF/p75/p52 molecule has both pro- and anti-apoptotic subdomains, the exact extent and function of which is only beginning to be unraveled. One strategy for improved chemotherapy of cancer cells with overexpression of LEDGF/p75 can be to remove the anti-apoptotic function inherent in the residues coded by exon 6, by first studying the effect of abolishing the NLS and the AT-hook present in this region.
AML blasts were collected from the peripheral blood of 13 patients. Blasts were collected from 3 patients with chemo-sensitive AML at the time of diagnosis ("low risk", L1-3). All had complete hematological remission after the first induction cycle. Two are still alive 93 and 109 months after diagnosis, respectively; the third had 44 months of disease-free survival even though consolidation therapy could not be completed due to serious side effects. All three had low/intermediate risk of relapse [4, 31] judged from cytogenetics (two inv(16), one normal) and no genetic Flt3 abnormality. AML cells were collected from five patients who had undergone previous intensive therapy, (i) two patients had relapse after intensive chemotherapy and allotransplantation, respectively, and (ii) three patients had primary resistant disease and did not reach remission after three induction cycles. For comparison, cells were collected at the time of primary diagnosis from patients with high risk of resistance/relapse [31–33]. AML ("high-risk", H1-5). Two patients (H1, 2) had AML secondary to primary myelodysplastic syndrome (MDS), and treatment was not attempted, whereas the three last patients (H3-5) later showed resistance to at least two different induction regimens. All human samples were collected in accordance with the Helsinki Declaration.
Preparation of native AML blasts
Leukemic peripheral blood mononuclear cells (PBMC) were collected from peripheral blood, and isolated by density gradient separation (Ficoll-Hypaque; NyCoMed, Oslo, Norway; specific density 1.077) [34, 35]. Cells were stored in liquid nitrogen until use. The percentage of blasts among leukemic PBMC exceeded 95% for all patients, as judged by light microscopy of May-Grünwald-Giemsa stained smears.
Culturing, transfection and apoptosis scoring in cell lines
Flp-In 293 cells (Invitrogen) were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum. The p52 specific splice variants were cloned into pcDNA3.1/V5-His TOPO (Invitrogen). LEDGF/p75 in pcDNA3.1/His was a generous gift from C. A. Casiano (St. Loma Linda University, USA). Cells were cultured and transfected using FuGENE-6 (Boehringer Mannheim) according to the manufacturer's protocol. After 24 h, cells were diluted to desired number and seeded into collagen coated 12-well plates. Cells were fixed by 4% buffered paraformaldehyde after DNR treatments. Cells were examined for daunorubicin-induced apoptosis by fluorescence microscopy after DNA staining [16, 34]. For estimation of statistical significance of values for percentage of apoptosis, we used the non-parametric Wilcoxon paired comparison test.
Cell culturing, virus production and assessment of apoptosis
The promyelocytic leukemia cell line IPC-81 and NB4 cells were generous gifts from Dr M Lanotte (INSERM U-496, Centre G Hayem, Hospital St Louis, Paris, France). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) under standard conditions supplemented with 10% heat-inactivated horse serum (Invitrogen), streptomycin (5 μg/ml) and penicillin (5 U/ml). Cells were kept in logarithmic growth, and as a standard procedure, cell density was adjusted to 0.2 × 106 cells/ml before addition of drugs. The cyclic AMP analog 8-chlorophenylthio-cAMP (8-CPT-cAMP) was purchased from Biolog Life Science, Bremen, Germany. Daunorubicin (DNR) was purchased from Sigma-Aldrich Inc. (St Louis, MO, USA).
Phoenix-Eco virus producer cells and CRU5-IRES-GFP retrovirus vector were kindly provided by Dr. J. Lorens (Department of Biomedicine, University of Bergen) and the cells were maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum (Invitrogen). The full-length cDNAs of LEDGF/p75 and p52b with Bst XI linker were cloned into CRU5-IRES-GFP.
Retrovirus were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG) and concentrated by ultracentrifugation essentially as described . Briefly, 1.5 × 106 Phoenix cells plated the day prior to transfection using the calcium phosphate method, 2 μg retroviral plasmid and 2 μg VSV-G. After 48 hours, filtered supernatants were concentrated by centrifugation for 3 hours at 50 000 g, 4°C. IPC-81 cells (0.5 × 106) were transduced by spin infection in which a mixture of virus and cells was centrifuged at 1200 g for 90 minutes at room temperature in the presence of protamine sulphate (5 mg/ml, Sigma-Aldrich). Routinely, 90–95% of the cells showed fluorescence 48 hours after infection. After treatment, cells were fixed in phosphate-buffered saline containing 2% formaldehyde. Screening for apoptosis was done blindly by two independent experienced evaluators using phase contrast microscopy to study surface morphology and nuclear morphology as visualised by the DNA-specific stain bisbenzimide, Hoechst 33342 (Sigma-Aldrich). Three different fields were randomly selected for counting at least 300 cells. All experiments were repeated at least five times. Percent apoptosis is defined as the ratio between cells with both membrane budding and chromatin condensation and the total of cells counted.
Differential screening of a leukemia cDNA library
The construction of a leukemia cDNA library and hybridization was as described previously . The SMART system (Clontech) was used to construct a cDNA library from the pooled RNA collected from AML patient cells (Table 1; S1 to S3 and R1 to R5). The UV-cross linked replica membranes with total over 25,000 colonies were subjected to primary screening by radio-labeled first strand cDNA from the pooled RNA of either "low risk" (L1-3) or "resistant" (R1-5) AML cells. Colonies with different signal intensity towards cDNA derived from resistant and sensitive AML were subjected to a second round of screening. DNA sequence reactions were carried out by the BigDye terminator method and analyzed by automatic sequencing (ABI PRISM). The BLAST search program was used for homology search (National Center for Biotechnology Information, Bethesda, MD).
Dot-blot analysis of differentially expressed genes in cells from individual AML patients
Gene fragments from selected differentially expressed genes were amplified from plasmids by PCR using T7 promoter and M13 reverse primers. The amplified specific PCR fragments were used in dot-blot analysis using 32P-labeled cDNA probes from RNA of L1, 2, 3; R1, 2, 3, 4, 5 and H1, 2, 3, 4, and 5. The membranes were analyzed by phospho-imaging, and the hybridization signal normalized to the mRNA expression level of the ribosomal protein P2 (rpP2) gene for sample from each AML patient.
The 5'-end of ARAP mRNA was obtained by using 5'Generacer kit (Invitrogen) with primers described in the kit and antisense primers designed from the cDNA sequence. The antisense primers, 5'-GTCATCATCTTCTGAGTCTGACTC-3' and 5'-CACCATCATCACCTTGTTTTTGCC-3', were used for PCR amplification.
Relative mRNA expression determined by RT-PCR
Total RNA from either NB4 cells or from patient AML blasts pooled from S1-3 or R1-5 was used for ss cDNA synthesis using oligo-(dT)-primer and superscript II reverse transcriptase (Invitrogen). The PCR reaction (50 μl) contained 1 μl cDNA and specific 5'-and 3'-primers. The PCR products were collected after 21–24 cycles of amplification. The primers were: rpL6, 5'-ATGGCGGGTGAAAAAGTTGAGAAGC-3' and 5'-GTAGAACACCAATTTGTGAGGA-3'; rpS4, 5'-ATGGCTCGTGGTCCCAAGAAGCATC-3' and 5'-GCACCCACTGCTCTGTTTGGCCGCC-3'; ARAP, 5'-CAAAAACAAGGTGATGATGATGG-3' and 5'-CCAGAGGGGCGGCAGCAGTCC-3'; Primers for full-length ARAP, 5'-ATGCCCAAATCCAAGCGCGACAA-3' and 5'-GTCATCATCTTCTGAGTCTGACTC-3'; LEDGF/p75, 5'-ATGACTCGCGATTTCAAACCTGGA-3' and 5'-GTTATCTAGTGTAGAATCCTTCAGA-3'; p52, 5'-ATGACTCGCGATTTCAAACCTGGA-3' and 5'-GTCCAATGAGTCTGTATCAAGATC-3'; p75-1, 5'-ATGACTCGCGATTTCAAACCTGGA-3' and 5'-GATCTTCATCTCTTGTTTGCTCCAC-3'; P75-2, 5'-AAGCCACCCACAAACAAACTACCC-3' and 5'-CCCTCCTTTTCTCTTCTTTTCTCC-3'; rpP2, 5'-ATGCGCTACGTCGCC-3' and 5'-TTAATCAAAAAGGCCAAATCCCAT-3'.
Western blot analysis
Cell pellets were lyzed in ice-cold 10 mM Tris pH 7.5 with 0.5% NP-40, 1 mM MgCl2, 1 mM DTT, 10 mM KCl, 0.4 M NaCl for 5 min, and centrifuged at 11,000× g for 5 min. 50 μg supernatant protein was separated by 12% SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to a nitrocellulose filter. The membrane was probed with anti-LEDGF/p75 polyclonal antibodies (kindly provided by C. A. Casiano) and the membrane was developed using the enhanced ECL detection method (Amersham Biosciences, Uppsala, Sweden).
acute myelogenous leukemia
A ML, r esistance a ssociated p rotein
Lens Epithelial Derived Growth Factor
EGF response factor 2
leukemic peripheral blood mononuclear cells
This work is part of the Cancer Locus, Medical faculty, University of Bergen initiative. We thank members of the Cancer Locus, University of Bergen, for their help and discussion during this work. The excellent technical assistance of Nina L. Larsen is highly appreciated. This work was supported by the Norwegian Cancer Society (Kreftforeningen) through grants to B.T.G., Ø.B., S.O.D., and J.R.L.
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