Oncogenic transformation of mesenchymal stem cells decreases Nrf2 expression favoring in vivo tumor growth and poorer survival
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The transcription factor Nrf2 is a key regulator of the cellular antioxidant response, and its activation by chemoprotective agents has been proposed as a potential strategy to prevent cancer. However, activating mutations in the Nrf2 pathway have been found to promote tumorigenesis in certain models. Therefore, the role of Nrf2 in cancer remains contentious.
We employed a well-characterized model of stepwise human mesenchymal stem cell (MSC) transformation and breast cancer cell lines to investigate oxidative stress and the role of Nrf2 during tumorigenesis. The Nrf2 pathway was studied by microarray analyses, qRT-PCR, and western-blotting. To assess the contribution of Nrf2 to transformation, we established tumor xenografts with transformed MSC expressing Nrf2 (n = 6 mice per group). Expression and survival data for Nrf2 in different cancers were obtained from GEO and TCGA databases. All statistical tests were two-sided.
We found an accumulation of reactive oxygen species during MSC transformation that correlated with the transcriptional down-regulation of antioxidants and Nrf2-downstream genes. Nrf2 was repressed in transformed MSC and in breast cancer cells via oncogene-induced activation of the RAS/RAF/ERK pathway. Furthermore, restoration of Nrf2 function in transformed cells decreased reactive oxygen species and impaired in vivo tumor growth (P = 0.001) by mechanisms that included sensitization to apoptosis, and a decreased hypoxic/angiogenic response through HIF-1α destabilization and VEGFA repression. Microarray analyses showed down-regulation of Nrf2 in a panel of human tumors and, strikingly, low Nrf2 expression correlated with poorer survival in patients with melanoma (P = 0.0341), kidney (P = 0.0203) and prostate (P = 0.00279) cancers.
Our data indicate that oncogene-induced Nrf2 repression is an adaptive response for certain cancers to acquire a pro-oxidant state that favors cell survival and in vivo tumor growth.
KeywordsAntioxidants Nrf2 oncogenes ROS survival HIF-1α
Mesenchymal stem cell
Human mammary epithelial cells
Human umbilical vein endothelial cells
Reactive oxygen species
Gene set enrichment analysis
Antioxidant response element
The cancer genome atlas
Gene expression omnibus
Kidney renal clear cell carcinoma
Skin cutaneous melanoma.
An increase in reactive oxygen species (ROS) is a common biochemical property of cancer cells[1, 2]. However, excess ROS also induce senescence, cell cycle arrest and apoptosis, indicating that redox homeostasis is tightly regulated in tumor cells. To offset excess ROS cells have developed regulatory mechanisms, including the induction of antioxidant enzymes and/or the activation of redox buffering systems such as glutathione. The transcription factor Nrf2 (NFE2L2) plays a crucial role in the cellular defense against oxidative stress through its ability to induce the expression of antioxidant and detoxification genes[4, 5]. Under basal conditions, Nrf2 is bound to its inhibitor Keap1 and targeted for degradation by the proteasome pathway[6, 7]. Upon certain stress conditions, Nrf2 is released from the inhibitory complex and translocates to the nucleus where it binds antioxidant response elements (ARE) in the promoter regions of its target genes[8, 9]. Among these genes are NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), members of the glutathione S-transferase (GST) family and genes involved in NADPH generation and glutathione biosynthesis[5, 10, 11, 12, 13].
Activation of the Nrf2-ARE pathway has been proposed as a potential strategy to prevent cancer because of its ability to suppress genotoxic insults by inducing antioxidants and detoxifying enzymes[14, 15, 16]. In this regard, nrf2-/- mice are more susceptible to chemically-induced cancer[17, 18, 19, 20], and Nrf2-deficiency has been suggested to favor metastasis. However, Nrf2 activation has also been proposed to play a role in cancer evolution[22, 23, 24, 25, 26], and induction of Nrf2 pathway due to genetic variants in Keap1 or Nrf2 might predispose to cancer[27, 28, 29, 30]. Therefore, the role of Nrf2 in cancer is contentious.
Here we employed a previously well-characterized model of human mesenchymal stem cell (MSC) stepwise transformation to mechanistically investigate changes in ROS levels during tumorigenesis. We found an accumulation of ROS during MSC transformation that correlated with the transcriptional down-regulation of antioxidants and ARE-containing genes. Moreover, Nrf2 expression was repressed in transformed MSC and breast cancer cells via activation of RAS/RAF/ERK pathway, and restoration of Nrf2 levels in transformed MSC induced the cellular antioxidant response and impaired in vivo tumor growth through mechanisms involving sensitization to apoptosis and destabilization of HIF-1α. Microarray comparison studies showed that expression of Nrf2 is down-regulated in a panel of human tumors, and lower expression of Nrf2 is associated with a poorer outcome in patients with melanoma, kidney and prostate cancers. Overall our results indicate that defects in the cellular antioxidant capacity contribute to ROS accumulation during transformation, and that oncogene-induced Nrf2 repression is an adaptive response for certain cancer cells to acquire a pro-oxidant state that favors cell survival and tumor growth.
In vitro transformation of human MSC leads to an increase in intracellular ROS that contributes to the transformed phenotype
Transformation of MSC induces transcriptional down-regulation of antioxidant genes
We focused on the last steps during MSC transformation where significant changes in intracellular ROS levels were found (from MSC3 to MSC4 and tMSC). qRT-PCR experiments confirmed down-regulation of Nrf2 and selected antioxidants and ARE-containing genes when tMSC were compared with MSC3 and MSC4 (Figure 2C). One of the most powerful antioxidants and a major redox buffering mechanism in the cell is the glutathione system (GSH/GSSG). Expression of genes involved in glutathione biosynthesis such as glutamate-cysteine ligase catalytic and modifier subunits (GCLC and GCLM), and glutathione synthetase (GSS) fluctuated during the process of MSC transformation (Figure 2D). We also found diminished expression of glutathione reductase (GSR, another Nrf2-down-stream gene) in tMSC, suggesting that inefficient conversion of oxidized glutathione (GSSG) to its reduced form (GSH) occurs in tumor cells (Figure 2D). Concurring with these results, tMSC showed the lowest levels of the active (reduced) form of glutathione (GSH), the form of glutathione able to provide antioxidant power (Figure 2E). Overall, these data indicate that transformation of MSC leads to a global transcriptional down-regulation of the cellular antioxidant program.
Nrf2 is repressed during cellular transformation via activation of RAS/RAF/ERK pathway
Recent reports showed that Nrf2 expression was decreased in certain human breast cancer cells and breast tumors when compared with normal mammary epithelial cells or normal breast tissue[36, 37]. Interestingly, we found a reduction in Nrf2 and NQO1 expression when normal human mammary epithelial cells (HMEC) were transformed using the same oncogenic elements that we employed to transform MSC (Figure 3C), suggesting that this mechanism for Nrf2 regulation is not restricted to adult MSC. Next we used chemical inhibitors to address whether Nrf2 expression is transcriptionally regulated via ERK or PI3K/AKT pathways in the breast cancer cell lines MDA-MB-231 and MCF-7. While cell survival was not affected by the concentration of inhibitors used in this assay (Additional file2: Figure S1), treatment with the ERK inhibitor U0126 led to a significant increase in the transcription of Nrf2 and NQO1 (Figure 3D). However, inhibition of AKT with GSK690693, or PI3K with LY294002 and wortmannin did not induce expression of Nrf2 nor NQO1 (Figure 3D). The effect of these inhibitors on ERK and PI3K/AKT pathways is shown in Figure 3E, where a modest but consistent activation of the Nrf2 pathway could be detected following only 16 hours treatment with U0126. Overall our data indicate that the RAS/RAF/ERK pathway mediates Nrf2 repression in these cancer cells.
Nrf2 activity was found suppressed in tumor cells due to increased expression of the ubiquitin ligase Cul3 that, together with Keap1, targets Nrf2 for degradation by the proteasome. However, expression of Keap1 (which is wild type in MSC, data not shown) and Cul3 did not increase in transformed MSC (Additional file3: Figure S2).
Nrf2 protein stabilization by means of tert-butylhydroquinone (TBHQ) impairs MSC transformation
Restoration of Nrf2 expression in tMSC induces the cellular antioxidant response and impairs in vivo tumor growth
Nrf2 over-expression sensitizes tMSC to apoptosis and diminishes the angiogenic response by destabilization of HIF-1α and VEGF repression
ROS are implicated in the response to hypoxia through a mechanism involving stabilization of hypoxia-inducible factor 1 (HIF-1α). Interestingly, tMSC over-expressing Nrf2 were not able to stabilize HIF-1α at 1% O2 concentration (Figure 6D). Furthermore, the expression of vascular endothelial growth factor A (VEGFA), an angiogenic HIF-1α-downstream gene, was significantly reduced in Nrf2-expressing cells grown at 21% O2 (Figure 6E). VEGFA production was further decreased when Nrf2-expressing cells were grown at 5% and 1% O2 concentrations (Figure 6E). Besides, we also found that cells over-expressing Nrf2 in hypoxic conditions showed a significant decreased expression of adrenomedullin (ADM), another HIF-1α-dependent angiogenic and anti-apoptotic gene (Additional file7: Figure S6).
Angiogenesis depends on the capacity of endothelial cells to proliferate and migrate. We next tested whether viability of human umbilical vein endothelial cells (HUVEC) is affected by conditioned medium from transformed cells over-expressing Nrf2. HUVEC cultured with hypoxic conditioned medium from tMSC expressing Nrf2 showed a significant impairment in viability when compared with HUVEC treated with hypoxic conditioned medium from tMSC expressing empty vector (Figure 6F). This result suggests that loss of Nrf2 expression in tumor cells could facilitate the proliferation of endothelial cells within the tumor microenvironment in conditions when oxygen concentration becomes limited.
Lower Nrf2 expression is associated with poorer survival in certain cancers
Next we investigated whether Nrf2 levels are associated with survival in patients with cancer. Analysis of available survival datasets obtained from GEO and TCGA databases showed that lower expression of Nrf2 (NFE2L2) is associated with a significantly poorer outcome in skin cutaneous melanoma (SKCM) (P = 0.0341) and in kidney clear cell carcinoma (KIRC) (P = 0.0203) (Figure 7B). Similarly, low Nrf2 expression was associated with biochemical recurrence in prostate cancer (PRAD-GSE21034) (P = 0.00279) (Figure 7B), but we found no relation positive or negative to prognosis in any of the other cancers studied (detailed in Additional file11: Table S2). The analysis of those cancers where we found an association between Nrf2 expression and survival revealed that the mRNA level of Nrf2 was positively correlated to its downstream targets in KIRC and PRAD-GSE21034, but not in SKCM (Figure 7C). These data suggest that the Nrf2 pathway activity can be diminished in those tumors exhibiting low Nrf2 expression.
Intracellular redox homeostasis is altered in cancer, where increased levels of ROS favor a pro-oxidant microenvironment. Here we show that MSC accumulate ROS during oncogenic transformation, and that transformed MSC become oxidative stress-dependent, since treatment with antioxidants decreases ROS levels and impairs their tumorigenic potential. Moreover, the increase in ROS coincides with the down-regulation of genes involved in the cellular antioxidant machinery, including most antioxidant enzymes, genes implicated in glutathione homeostasis, and those involved in the biosynthesis of NADPH. It is believed that a significant amount of the intracellular ROS is produced by mitochondria. However, ROS can also be produced by non-mitochondrial sources such as membrane-bound NADPH oxidases. While the vast majority of the pro-oxidant enzymes in our list of ROS genes (including most of the NADPH oxidases) do not change during MSC transformation (data not shown), microarray and qRT-PCR analysis showed increased expression of NADPH oxidase 4 (NOX4) and aldehyde oxidase 1 (AOX1) during MSC transformation (data not shown). Although the precise contribution of these enzymes to ROS accumulation is unknown and needs further investigation, our data overall suggest that a defective cellular antioxidant system may largely contribute to the high levels of ROS observed during MSC transformation.
We also found that expression of Nrf2 decreased during the process of MSC transformation. Although we cannot rule out the possibility that both ST and oncogenic Ras may interfere with Nrf2 protein stability, we focused our attention on the role of H-RasV12 as it induced the most marked down-regulation of Nrf2 expression. Our data indicate that activation of the RAS/RAF/ERK pathway represses Nrf2 expression and contributes to the diminution of the cellular antioxidant response during MSC transformation. Nrf2 and its downstream target NQO1 were also suppressed in transformed human mammary epithelial cells, indicating that this mechanism for ROS accumulation is not restricted to adult stem cells. These results are in concordance with previous reports where ERK inhibition in the presence of insulin increases ARE-luciferase activity in HL-1 mouse cardiac cells, and where the RAS/RAF/ERK pathway was proposed to inhibit Nrf2 in human neuroblastoma cells with Myc amplification. Moreover, analysis of previous microarray studies where investigators have transformed cells in vitro[47, 48] showed that oncogenic transformation leads to Nrf2 down-regulation in both mouse and human cells (Additional file12: Figure S10). However, our results are in contrast to those from a report by DeNicola et al. where conditional activation of K-RasG12D in a mouse model of pancreatic cancer induced the expression of Nrf2 via the RAF pathway. This divergence could be due to the different approach employed to express oncogenes, as H-RasV12 was constitutively expressed in human MSC and breast epithelial cells, whereas K-RasG12D was conditionally activated in the mouse model. These approaches might elicit quantitative different levels of Ras activity in the target cells, resulting in a different regulatory mechanism for Nrf2 expression. However, rather than super-physiologic expression of Nrf2, we restored Nrf2 levels to that observed in non-transformed MSC, suggesting that our model is relevant to transformation of primary human cells. Other divergences between our work and that from DeNicola et al. are the different species and tumor models studied, as well as the different stage during tumor development. In this regard, oncogenic Ras might induce different biological responses depending on the status of tumor suppressors such as p53 and/or oncogenes such as Myc.
Here we show that Nrf2-mediated induction of the cellular antioxidant response is an efficient strategy to tackle in vivo tumor growth in transformed adult stem cells. Mechanistically, we show that Nrf2 sensitizes transformed cells to apoptosis, contrasting with previous reports where Nrf2 was shown to protect from apoptosis and to enhance drug resistance[49, 50]. However, our results are in concordance with previous findings where the presence of antioxidants was found to improve the cytotoxic effect of apoptosis-inducing agents. Future studies should address the effects of Nrf2 on the regulation of pro- and anti-apoptotic proteins in transformed MSC.
We also provide evidence linking Nrf2 activation with a reduced angiogenic response under hypoxic conditions. In agreement with findings that ROS may regulate angiogenesis and tumor growth through HIF-1α and VEGF, over-expression of Nrf2 in tMSC led to a diminished hypoxic response through destabilization of HIF-1α and reduced VEGFA and ADM expression. These data differ from a report where Nrf2 knockdown by siRNA in human colon cancer cells inhibited tumor growth and led to a reduction in VEGF expression. However, our data suggest that hypoxic conditions could result in a more hostile microenvironment for cells with higher levels of Nrf2.
All these discrepancies add more complexity to the contentious function of Nrf2 during tumorigenesis. Indeed, it has been suggested that the role of Nrf2 in cancer is context-dependent. In this regard, a recent report based on an urethane-induced multistep mouse model of lung cancer has proposed that Nrf2 has the dual role of preventing tumor initiation, but also promoting tumor progression. However our data reveal a tumor suppressor role for Nrf2 since its down-regulation contributes to cellular transformation and in vivo tumor growth. Microarray comparison studies support our experimental data, indicating that expression of Nrf2 is down-regulated in many tumors. Moreover, analysis of available survival datasets obtained from GEO and TCGA databases shows that increased Nrf2 expression correlates with better survival in patients with melanoma, kidney and prostate cancers, further supporting our in vivo findings where restoration of Nrf2 expression in transformed MSC improved survival.
Overall our results indicate that defects in the cellular antioxidant capacity contribute to ROS accumulation during transformation, and that oncogene-induced Nrf2 repression is an adaptive response for certain cancer cells that favors in vivo tumor expansion and poorer survival. We also show that rescue of Nrf2 function in fully transformed cells is an effective strategy to tackle in vivo tumor growth, as Nrf2 expression sensitizes transformed cells to apoptosis and impairs the angiogenic response through destabilization of HIF-1α.
Cell culture and generation of stable cell lines
Culture conditions, retrovirus production and generation of cell lines were previously described. Briefly, primary human MSC previously isolated from the bone marrow of a healthy donor (MSC0) according to institutional guidelines were serially transduced with retroviruses encoding hTERT (MSC1), E6 and E7 from HPV-16 (MSC3), ST antigen from SV40 (MSC4), and H-RasV12 (tMSC). For the generation of tMSC over-expressing Nrf2, we amplified the Nrf2 gene from human cDNA using the following primers: forward (5′-GCGGATCCATGATGGACTTG-3′) and reverse (5′-ACGCGTCGACCTAGTTTTTCTTAACATC-3′). Nrf2 was later cloned into pWZL-hygro and used to infect tMSC where H-RasV12 had been previously introduced with pWZL-blast.
Detection of intracellular ROS
ROS levels were quantified by staining the cells with MitoSOX Red and CM-H2DCFDA dyes (both from Invitrogen, Paisley, UK). After 30 minutes incubation with the dyes at 5 μM final concentration, cells were collected and analyzed by flow cytometry using either a FACSCalibur instrument (BD Biosciences, San Jose, CA) or a CyAN flow cytometer (DAKO, Cambridgeshire, UK). Data were analyzed using either CellQuest V or Summit software. Cell incubation with MitoSOX was performed in serum-depleted media.
Soft agarose colony formation by anchorage-independent growth and tumor xenografts were previously described. The animal experiments were conducted in accordance with institutional guidelines under the approved protocols. For the in vivo tumor growth experiments, Kaplan Meier survival plots were generated, and from the survival data a log rank (Mantel-Cox) test was used to demonstrate significant differences between groups.
Antibodies and reagents
The following antibodies were used for immunoblotting: rabbit polyclonal C-20 (Santa Cruz, CA), mouse monoclonal (M01) clone 1 F3 (Abnova, Taiwan), and rabbit monoclonal EP1808Y (Abcam, Cambridge, UK) for Nrf2; Actin was from Calbiochem/MerckMillipore (Watford, UK); NQO1 was from Novus Biologicals (Cambridge, UK); G6PD was from Bethyl (Montgomery, TX); HIF-1α was from BD Biosciences (San Jose, CA); Cleaved PARP, total AKT, phosphorylated AKT (Ser473) (p-AKT), total ERK1/2, phosphorylated ERK1/2 (Thr202/Tyr204) (p-ERK), Cul3, Keap1, HSP90 and Lamin A/C antibodies were all from Cell Signaling Technology (Danvers, MA); GAPDH was from Advanced Immunochemical Inc. (Long Beach, CA); Secondary antibodies were from DAKO.
N-acetyl-L-cysteine, ascorbic acid, tert-butylhydroquinone, camptothecin, etoposide and staurosporine were all obtained from Sigma (Dorset, UK).
Apoptosis was induced by treatment with 5 μM camptothecin for 24 hours, 1 μM etoposide for 48 hours, and 1 μM staurosporine for 3 hours. The percentage of apoptotic cells was measured by flow cytometry after double staining with Annexin-V (FITC) and Propidium Iodide (PI) using the FITC Annexin-V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA) following the manufacturer’s instructions. Data were analyzed using Summit software. Caspase 3/7 activity was quantified by using Caspase-Glo 3/7 Assay from Promega (Southampton, UK). Cell viability was addressed by using CellTiter AQueousOne Solution Cell Proliferation Assay (Promega), a colorimetric method based on the reduction of a tetrazolium compound by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. Levels of reduced glutathione (GSH) were quantified by using GSH-Glo Glutathione Assay (Promega) following the manufacturer’s instructions. Nuclear and cytoplasmic protein fractions were obtained by using NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Cramlington, UK). Experiments in hypoxia (1% or 5% oxygen concentration) were performed as previously described.
In the inhibition studies for the RAS-downstream signaling pathways, breast cancer cell lines MDA-MB-231 and MCF-7 were seeded onto 6-well plates and 24 hours later washed with PBS and subjected to free serum standard media. 24 hours later the cells were incubated with free serum standard media containing DMSO (Sigma) or the following chemicals: ERK kinases inhibitor U0126 (Calbiochem/MerckMillipore, Watford, UK); PI3K inhibitors LY294002 (Merck Biosciences Ltd, Nottingham, UK) and wortmannin (Cell Signaling Technology, Danvers, MA); and AKT inhibitor GSK690693 (Symansis, Timaru, NZ). After 16 hours incubation, RNA was collected and qRT-PCR was performed. Protein extracts were also collected for western-blot analysis.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using RNEasy mini kit (Qiagen, Sussex, UK) and mRNA levels were quantified by qRT-PCR using Taqman Gene Expression Assays (Applied Biosystems, Paisley, UK). SYBR Green Master Mix (Applied Biosystems) was used with optimized forward (5′-GGAGTCAACGGATTTGGTCGTA-3′) and reverse (5′-GGCAACAATATCCACTTTACCAGAGT-3′) primers for GAPDH (reference gene) at final concentration of 300 nM. b-Actin (Applied Biosystems) was used as reference gene for the experiments in hypoxia. All experiments were performed at least by triplicate.
Additional cell lines
Additional MSC lines with activated RAS or RAS-downstream effectors were generated by infection of MSC4 with the retroviral vector pBabe-hygro encoding constitutive active RAS (H-RasV12), the membrane-targeted RAF-1 (Raf-CAAX), and myristoylated AKT (myrAKT), all kindly provided by Dr. Pablo Rodriguez-Viciana (UCL Cancer Institute, London, UK). Immortal human mammary epithelial cells (HMEC), obtained from Dr. Rodriguez-Viciana, were cultured in DMEM/F-12 containing 5% horse serum (Life Technologies/Invitrogen, Paisley, UK) and supplemented with EGF (20 ng/ml) (Peprotech, London, UK), hydrocortisone (500 ng/ml), cholera toxin (100 ng/ml) and insulin (10 μg/ml), all from Sigma (Dorset, UK). HMEC expressing different oncogene combinations were generated after infection with the same retroviral vectors used for the generation of MSC lines. Breast cancer cell lines MDA-MB-231 and MCF-7 were cultured in DMEM containing 10% fetal bovine serum (Life Technologies/Invitrogen). Human umbilical vein endothelial cells (HUVEC) were obtained from Promocell (Heidelberg, Germany) and cultured with Endothelial Cell Growth Medium (Promocell) according to supplier’s instructions.
Public transcriptome data
The expression level of Nrf2 in many types of cancer was compared using the Oncomine (https://www.oncomine.org/resource/login.html) and The Cancer Genome Atlas (TCGA) (https://genome-cancer.ucsc.edu) databases of cancer expression data. We downloaded the available expression data with clinical details for 15 types of cancer from TCGA (https://tcga-data.nci.nih.gov/tcga) and 3 types of cancer from the NCBI gene expression omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo), including 5 separate breast cancer study datasets. In total, for survival analyses we studied 16 distinct types of cancer. Details of each dataset, the number of samples with clinical details, the expression platform, and associated Pubmed IDs for the GEO datasets are in Additional file13: Table S3.
Gene expression microarray analysis
Generation of Gene Expression Microarrays (GEM) was previously described and data were deposited in ArrayExpress database (accession no. E-MEXP-563). Gene Set Enrichment Analysis (GSEA) measures the enrichment of a gene set within a GEM experiment. The enrichment score (ES) is a metric of the skew of a gene set within the rank of genes sorted by their GEM expression difference. The significance of enrichment (q, or false discovery rate) is the proportion of true ES >1000 ES generated from random gene sets (of equal size) (e.g., proportion of ESOBSERVED > ESNULL). Leading-edge genes are the subset that contributes most to the ES.
For survival analysis we used the R survival package. To survey for potential association between gene expression and survival we categorized samples as below or above median expression for each gene and then calculated the log-rank (Mantel Cox) P value comparison between the groups. For KIRC, SKCM and PRAD-GSE21034 datasets with significant NFE2L2 log rank tests we also calculated the hazard ratio using the Cox proportional hazard model. Elsewhere data were analyzed using Student’s t test, Spearmans rho or log-rank test as appropriate for the analysis. Values are given as mean ± SD. All statistical tests were two-sided, and results were considered statistically significant when P < 0.05.
We thank F. Gratrix, Dr. O. Yogev and Dr. L. Nikitenko for technical assistance and very helpful discussions, Dr. Pablo Rodriguez-Viciana for providing retroviral constructs and cell lines, and Dr. J. Martin-Serrano for providing retroviral packaging plasmids.
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