Expression of KDR and FLT4 in leukemia and lymphoma cell lines
The VEGF-Rs KDR
) and FLT4
) are not only expressed on blood endothelial and lymphendothelial cells, but also on solid tumors and leukemias. Leukemia-derived VEGFs may induce the growth of leukemic cells in an autocrine or paracrine fashion [7
]. The promoters of VEGF-Rs and their ligands contain CpG islands, regulatory regions that are typically methylated in epigenetically silenced genes [14
]. Recent reports show that expression of FLT1
are controlled by promoter methylation [14
]. However, only a limited number of leukemia and lymphoma cell lines have been tested for VEGF-R
expression and promoter methylation hitherto.
To find model systems for VEGF-R regulation, we tested some ninety leukemia and lymphoma cell lines for KDR and FLT4 mRNA expression. Both genes were regularly expressed in leukemia but not in lymphoma cell lines: 10/62 (16%) cell lines from various leukemic entities expressed KDR, 27/65 (42%) expressed FLT4 (Table ). In contrast, 0/30 lymphoma cell lines expressed KDR, and only 1/30 (3%) expressed FLT4 (Table ). Cell lines with high VEGF-R transcript levels expressed also the corresponding proteins: cell lines CMK, HEL and MEG-01 expressed KDR, whereas cell lines HEL, MHH-CALL2, OCI-AML1 and SUP-B15 were FLT4 positive (Figure , Table ).
VEGF-R mRNA expression in leukemia and lymphoma cell lines
Figure 1 KDR and FLT4 in HDMECs, HUVECs and leukemia cell lines. KDR and FLT4 mRNA expression levels were determined by quantitative real-time PCR and indicated underneath the cell line name. TBP expression was used as endogenous control and cell lines CMK (KDR) (more ...)
Promoter methylation status and expression levels of KDR and FLT4
OCI-AML1: a model system for VEGF-C induced cell signaling
Cytokine-dependent cell lines have often and successfully been used as model systems for signal transduction studies. In contrast to primary cells, no contaminating cell fraction effects "false" signals in cell lines, and in contrast to cytokine-independently growing cell lines, cytokine starvation silences the relevant enzymes in cytokine-dependent cell lines. We chose cell line OCI-AML1 as this was the only cytokine dependent, FLT4
positive cell line tested (Table ). The cytokine response profile of this cell line has been published previously [20
]. Cell line OCI-AML1 did not show a proliferative response on VEGF-C (data not shown). However, short-term (5 min) stimulation with VEGF-C induced phosphorylation of ERK1/2 (Figure ). Preincubation with the FLT4 inhibitor MAZ51 inhibited this effect, confirming the specificity of the VEGF-C induced EKR1/2 activation (Figure ). ERK1/2 phosphorylation was tested because the p42/44 MAPK pathway is a known FLT4 target [21
]. The results of cell signaling experiments shown in Figure confirm that cell line OCI-AML1 is a model system for FLT4 signaling, expecially as KDR, the second receptor for VEGF-C is not expressed in this cell line (Table ).
Figure 2 Phosphorylation level of ERK1/2 in VEGF-C responsive OCI-AML1 cells. The cytokine-responsive cell line OCI-AML1 was cytokine-starved for 18 h, then stimulated for 5 min with VEGF-C. Pretreatment with the FLT4 inhibitor MAZ51 (20 μM, 1 h) prevented (more ...)
KDR: promoter methylation and gene expression
To test whether KDR is epigenetically regulated, we performed bisulfite sequencing of KDR negative and positive cell lines and of primary endothelial cells. The KDR negative cell line DOHH-2 had a highly methylated KDR promoter, the KDR positive cell line HEL was nearly unmethylated (Figure ). Largely unmethylated were also HDMECs and HUVECs, both expressing KDR (Figure ). To assess the KDR methylation status for a larger number of cell lines, we performed methylation-specific PCR (MSP), a technique less costly and laborious than bisulfite sequencing. The majority of KDR negative cell lines were methylated, KDR positive HUVECs were unmethylated (Figure ). However, even HDMECs were U-and M-PCR positive although they expressed high KDR levels and although only a small minority of clones were methylated according to sequencing analysis (Figures and ). Apparently, a low proportion of methylated CpGs was sufficient to yield signals in the M-PCR. The same was true for U-PCR: the KDR negative cell line DOHH-2 - highly methylated according to the results of bisulfite sequencing--showed signals in M- and in U-PCR (Figure , Table ).
Figure 3 Bisulfite sequencing of the KDR promoter. A CpG island is located between -1231 and 1125 relative to the ATG codon of KDR. The 3' part of the KDR promoter region and exon 1 (612 bp, 53 CpG sites) were sequenced after bisulfite conversion of DNA from cell (more ...)
Figure 4 Methylation status of KDR and FLT4 in cell lines and primary cells. The methylation status of KDR and FLT4 in leukemia cell lines and in HDMECs and HUVECs was analyzed by MSP after bisulfite conversion of the DNA. Agarose gels of KDR and FLT4 M- and U-PCR (more ...)
In spite of the high sensitivity--a certain drawback of the PCR-based MSP technique--the accuracy of KDR M-PCR was 88% supporting the notion that KDR expression is regulated by DNA methylation (Table ).
FLT4: promoter methylation and gene expression
Bisulfite sequencing and BSP analysis were also performed to analyze the methylation status of FLT4 in cell lines, HUVECs and HDMECs. Results of bisulfite sequencing showed that FLT4 was largely methylated in the FLT4 negative cell line EM-2 and unmethylated in the FLT4 positive cell line SUP-B15 as it was in HUVECs and HDMECs (Figure ). MSP analysis confirmed that FLT4 exhibited the inverse correlation between promoter methylation and gene expression that is indicative for epigenetic regulation (Figure , Table ). However, the accuracy of FLT4 M-PCR (80%) was lower than for KDR M-PCR (88%). Of note was also that TF-1 cells did not express FLT4 although the promoter was unmethylated (Table ). These data suggested that regulatory mechanisms other than DNA methylation are also important for the regulation of FLT4.
Figure 5 Bisulfite sequencing of the FLT4 promoter. A CpG island is located between -1231 and 769 relative to the ATG codon of FLT4. Part of the promoter region (337 bp, 20 CpG sites) was sequenced from cell lines EM-2 (FLT4 negative) and SUP-B15 (FLT4 positive) (more ...)
Effect of DNA demethylating agent 5-Aza-dC on expression of KDR and FLT4
To test whether KDR and FLT4 were silenced by promoter methylation, we treated methylated and unmethylated cell lines with the DNA demethylating agent 5-Aza-dC. In 4/5 KDR-negative cell lines, expression of KDR was induced by DNA demethylation (Table ). FLT4 expression was upregulated in 4/4 negative cell lines (Table ). KDR and FLT4 expression in positive cell lines were not affected (Table ). Although these results confirmed that promoter methylation plays a role for the regulation of these VEGF-Rs, we also noted substantial differences in the levels of 5-Aza-dC-triggered gene induction between different cell lines (Table ). Furthermore, even in the most sensitive cell lines (HL-60 for KDR induction, EM-2 for FLT4 induction), demethylation did not induce mRNA expression that would translate into protein levels detectable by Western blot analysis (data not shown). These results suggest that other mechanisms than DNA methylation are also involved in the regulation of KDR and FLT4.
Effect of 5-Aza-dC on expression of KDR and FLT4
Besides DNA methylation, also histone modifications are epigenetic mechanisms that affect the expression of individual genes. Just to mention two examples, acetylated histone H3 (at lysine 9 and 14) is a marker for gene activation [23
], tri-methylation of histone H3 lysine 27 stands for gene suppression [24
]. Furthermore, epigenetic modifications can influence each other: methylated CpGs in a promoter region can be targeted by proteins that interact with histone deacetylases. The consequence is an inactive chromatin status and transcriptional repression [25
However, besides epigenetic mechanisms, also the presence or absence of trans-acting factors may govern the expression of KDR
. Thus, it has been shown that transcription factor binding sites (Sp1, AP-2 and NFκB) are essential for the base-line activity of the KDR
]. Here, we set out to find whether NFκB also plays a role for the expression of FLT4
Influence of transactivating factors
During inflammation, new lymphatic vessels are formed. NF-κB is a key mediator of inflammatory processes and has recently been identified as inducer of FLT4
on lymphatic endothelial cells [13
]. To test whether NF-κB contributes to FLT4
expression in leukemic cells, we stimulated the FLT4
negative cell line EM-2 and the FLT4
positive cell line OCI-AML1 with synthetic MALP-2. MALP-2 binds to toll-like receptors-2 and -6 [28
]. MALP-2 triggers the NF-κB pathway [29
] which leads to the expression of NF-κB targets like TNFα
Accordingly, MALP-2 (100 ng/ml, 7 min) induced phosphorylation and degradation of the NF-κB inhibitor IκB and stimulated phosphorylation of p38 in cell lines EM-2 and OCI-AML1 (Figure ). MALP-2 (100 ng/ml, 1 h) triggered expression of the NK-κB targets TNFα (80× in EM-2, 1000× in OCI-AML1) and IP-10 (600× in EM-2, > 1000× in OCI-AML1) in both cell lines. However, the expression of FLT4 was not affected, neither in the FLT4 positive cell line OCI-AML1 nor in the FLT4 negative (methylated) cell line EM-2. MALP-2 did also not increase the FLT4 stimulating effect of 5-Aza-dC on EM-2 cells (data not shown). Thus, our results do not support the view that NF-κB is a transactivator of FLT4. We observed a 10-fold increase in KDR in cell lines OCI-AML1 and EM-2 (the latter pretreated with 5-Aza-dC). However, as the level reached after stimulation was still extremely low, it appears unlikey that NF-κB is an important regulator for KDR either.
Figure 6 Degradation of IκB and phosphorylation of p38MAPK in MALP-2 responsive cell lines. Cell lines EM-2 (FLT4 negative) and OCI-AML1 (FLT4 positive) were stimulated with MALP-2 (100 ng/ml). The NF-κB inhibitor IκB was phosphorylated (more ...)
KDR and FLT4 in HDMECs and HUVECs
HDMECs, HUVECs and KDR positive leukemia cell lines exhibited demethylated KDR promoters (Figure , Table ). However, mRNA and protein levels were distinctly higher in the primary cells than in the leukemia cell lines (Figure ). Results of Western blot analysis were confirmed by flow cytometry (Figure ). HDMECs and HUVECs expressed the pan-endothelial marker CD31 (Figure ). HDMECs, primarily consisting of lymphatic endothelial cells, were also positive for the lymphatic vessel marker podoplanin, HUVECs were podoplanin negative (Figure ). Both types of primary cells expressed much higher levels of KDR than KDR-positive cell lines (Figure ). Also FLT4 expression levels varied greatly from one cell type to the other: FLT4 expression of HUVECs was comparable to those of FLT4 positive cell lines, while HDMECs showed much higher FLT4 expression levels (Figures and ). These results are in line with our data of MSP analyses and 5-Aza-dC experiments suggesting that DNA methylation is not the only mechanism that controls KDR and FLT4 gene expression.
Figure 7 Expression of KDR and FLT4 on HDMECs and HUVECs. Flow cytometry analysis for KDR, FLT4, the lymphatic vessel marker podoplanin and the panendothelial marker CD31. Note that HDMECs and HUVECs show comparable KDR expression levels, while FLT4 is stronger (more ...)