The canonical Wnt pathway regulates several biological processes including development, cell growth and proliferation via consecutive gene regulation. A high number of target genes of the Wnt pathway ...has been identified, but the chronological order of target gene expression is still elusive. This order is supposed to be crucial for the controlled course of events downstream of the activated Wnt pathway. Here we present the expression chronologies of the target genes
Ccnd1 (encoding for cyclin D1),
Myc (c-Myc),
Cdkn1a (p21
CIP1/WAF1),
Tfrc (Transferrin receptor 1),
Plf1 (Proliferin-1) and
Ramp3 (Receptor activity-modifying protein 3) in C57MG cells after stimulation with Wnt-3a. We discriminated between immediate (below 1
h), early (between 1 and 6
h), intermediate (between 6 and 12
h) and late (after 12
h) targets. According to this classification
Myc and
Tfrc belong to the immediate target genes;
Ccnd1,
Plf1 and
Ramp3 are early target genes and
Cdkn1a is an intermediate target gene.
SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid ...leukemia (AML). The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene.
Of 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Ibeta)-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Ialpha-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.
Cell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.
CD7 is a negative prognostic marker in myeloid malignancies. In acute myeloid leukemia (AML), an inverse correlation exists between expression of wild-type CEBPA and CD7. Aim of this study was to ...find out whether C/EBPalpha is a negative regulator of CD7 and which other regulatory mechanisms might be involved.
As already described for primary AML cells, the majority of AML cell lines tested were either C/EBPalpha+/CD7- or C/EBPalpha-/CD7+. However, the existence of isolated CD7+ cell lines expressing wild-type C/EBPalpha challenges the notion that C/EBPalpha acts as a unique repressor of CD7. Furthermore, ectopic expression of CEBPA did not reduce CD7 in CD7+ cells and knock-down of C/EBPalpha failed to induce CD7 in CD7- cells. In contrast, the DNA demethylating agent Aza-2'deoxycytidine triggered CD7 expression in CD7- AML and in T-cell lines suggesting epigenetic regulation of CD7. Bisulfite sequencing data confirmed that CpGs in the CD7 exon1 region are methylated in CD7- cell lines, and unmethylated in CD7+ cell lines.
We confirmed an inverse correlation between the expression of wild-type CEBPA and of CD7 in AML cells. Our results contradict the hypothesis that C/EBPalpha acts as repressor for CD7, and instead show that epigenetic mechanisms are responsible for CD7 regulation, in AML cells as well as in T-cells, the typical CD7 expressing cell type.
Abstract 3469
Poster Board III-357
Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a common hallmark of cancer. It is generally agreed, that CpG ...island hypermethylation profiles are specific for different tumor types Costello et al., 2000, Nat Genet 25:132-138. Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification), methylation of 24 different TSG was analyzed in 40 lymphoma cell lines representing Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). On average 8 ± 2.8 TSG out of the 24 analyzed were methylated per lymphoma cell line, whereas 0/24 TSG were methylated in healthy donor tonsils. Methylation frequencies decreased from HL, ALCL, BL, FL, DLBCL to MCL cell lines. The TSG methylation status of the most relevant genes was verified by methylation-specific PCR. Moreover, TSG hypermethylation correlated with transcriptional silencing as assessed by quantitative real time PCR. While our studies on the methylation status of TSG in lymphoma cell lines support previous methylation analyses performed on primary lymphoma patient material for many of the genes analyzed here, MS-MLPA screening also revealed a new interesting candidate: CD44. Hypermethylation of CD44 was characteristic of ALCL, BL, FL and DLBCL cell lines and allowed their discrimination from MCL and HL cell lines. In CD44 hypermethylated cell lines expression of CD44 was re-inducible at mRNA and protein levels by treatment with the demethylating agent 5-Aza-2'-deoxycytidine, confirming its epigenetic regulation. Furthermore, CD44 ligation with an anti-CD44 antibody induced apoptosis in CD44+ (CD44 unmethylated) lymphoma cell lines whereas CD44 hypermethylated cell lines showed no response. Thus, CD44 might be an interesting new epigenetic marker and a potential molecular target for the diagnosis and treatment of specific lymphoma subtypes.
No relevant conflicts of interest to declare.
Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor ...prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines.
Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLLmu AML cells, while 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu AML cell lines. MLLwt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene.
These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.
Abstract 365▪▪This icon denotes an abstract that is clinically relevant.
Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed ...phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines.
Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene.
These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.
No relevant conflicts of interest to declare.
The methylation of CpG islands leads to the abnormal silencing of tumor suppressor genes and thus supposedly contributes to tumorigenesis. Recently, brain expressed X-linked-2 (BEX2) was described as ...candidate tumor suppressor gene in malignant glioma. With tissue expression array analyses, we could show that BEX2 was highly expressed in various brain-derived tissues, but not in hematopoetic cells of healthy donors. Also acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cell lines usually did not show high levels of BEX2. However, we found BEX2 highly expressed in those AML cell lines that carried aberrations of the mixed lineage leukemia gene (MLLmu). While MLL wild-type (MLLwt) cell lines showed hypermethylation of a CpG rich area within the BEX2 promoter, the BEX2 expressing, MLLmu cell lines did not show methylation of the BEX2 promoter. Confirming that BEX2 is an epigenetically regulated gene, aza-2′deoxycytidine increased the expression of BEX2 mRNA in MLLwt cells. Hypermethylation profiles of tumor suppressor genes may be used to identify subtypes of leukemia/lymphoma. To find out whether silencing and activation patterns of BEX2 in leukemia correlate with those of other tumor suppressor genes, we performed a polymerase-chain reaction (PCR) based assay (MLPA) that allowed us to simultaneously verify methylation- and ploidy status of 24 tumor suppressor genes. Twenty-eight human leukemia cell lines were tested, fourteen AML- and ALL-derived cell lines, half of them with MLL translocations. In summary, 7/24 tumor suppressor genes (plus BEX2) appear to be promising markers allowing for the distinction between ALL and AML, and between MLLmu and MLLwt AML. All AML and ALL cell lines were affected by methylation or deletion of CKDN2B and/or ESR1. Analysis of the methylation status of DAPK1 and FHIT allowed to distinguish between ALL and AML. The promoters of both genes, DAPK1 and FHIT were methylated in the majority of ALL cell lines: 11/14 ALL vs. 1/14 AML cell lines showed methylation of DAPK1, 13/14 ALL vs. 2/14 AML showed methylation of FHIT. There was no correlation between the methylation status of tumor suppressor genes and the MLL status in ALL cell lines. In contrast, MLLmu and wt AML cell lines showed differential promoter methylation patterns of IGSF4, RARB and TIMP3. At least two of these genes were methylated in every MLLwt AML cell line, but not in MLLmu AML. Quantitative real-time PCR demonstrated that expression of BEX2 and IGSF4 was silenced in cells carrying the methylated promoters. For RARB and TIMP3, additional mechanisms besides promoter methylation appear to be involved in gene regulation. These results show that methylation status of as few as four tumor suppressor genes (BEX2, IGSF4, RARB and TIMP3) may allow conclusions about the MLLmu/wt status of AML cells. Our results raise the question whether MLLmu proteins are directly or indirectly responsible for the hypomethylation of BEX2, IGSF4, RARB and TIMP3. If this is the case, tissue-specific effectors must play important additional roles as MLLmu ALL cells do not show this phenomenon.
The Wnt pathway controls biological processes via the regulation of target gene expression. The expression of direct Wnt target genes, e.g. cyclin D1and MYC, is activated by the transcription factor ...TCF, which binds to specific sequence motifs in the promoter. Indirect target genes are regulated via transcription regulators, which are targets of the Wnt pathway. As an example, MYC regulates the MYC interacting zinc finger protein‐1 (MIZ‐1), which is able to inhibit the expression of the indirect target p21WAF1. We intended to identify new Wnt target genes and to get a deeper insight into the regulatory mechanisms of Wnt target gene expression. For this we analyzed the differential expression pattern of Wnt‐1 activated cells by microarray analysis. We identified 43 sequences including eight expressed sequence tags (ESTs), which showed increased transcript levels, and 104 sequences including 19 ESTs with decreased RNA levels. Northern blot and real‐time quantitative PCR analysis of the differential expression levels of 15 genes confirmed the differential expression trends of eight candidate genes. When the Wnt pathway was regulated at the lower level of glycogen synthase kinase‐3β (GSK‐3β) or adenomatous polyposis coli (APC), we detected discrepant expression trends. We compared the number of binding sites of transcription factors in the genomic regions of all candidate target genes with the number of sites in control genes. We found that the genomic regions of the down‐regulated genes include an increased number of putative MIZ‐1 binding sites. Our study introduces several new Wnt target genes and provides indications that the specific gene expression pattern depends on the type of the activation trigger or the level of interference with the Wnt pathway. Furthermore, our data indicate that a high proportion of Wnt target genes are regulated by indirect mechanisms.
The first five years of the Wnt targetome Vlad, Annica; Röhrs, Sonja; Klein-Hitpass, Ludger ...
Cellular signalling,
20/May , Letnik:
20, Številka:
5
Journal Article
Recenzirano
The canonical Wnt pathway controls cell differentiation, proliferation and apoptosis by regulating the expression of a high number of target genes. The first target gene of the Wnt pathway was ...discovered nearly 20 years ago, when analysing gene expression patterns in the Drosophila embryo. Since the year 2002 entire transcriptomes have been screened by microarray analysis in order to identify genes, which are differentially expressed in cells with activated Wnt pathway. Recently, novel genome-based screening methods have been developed, which are less error-prone and independent from RNA. The exemplified methods STAGE (Sequence Tag Analysis Of Genomic Enrichment), ChIP-PET (chromatin immunoprecipitation with paired-end ditag), ChIP-Seq (ChIP followed by direct sequencing) and the bioinformatics approach EEL (Enhancer Element Locator) will be introduced shortly. The high number of potential target genes and regulated functions left questions unanswered, for instance how the Wnt pathway controls such a high number of genes and how it is able to regulate so many different cellular functions. In order to answer these questions we ordered the genes of the published Wnt target screenings according to their functions, and summarized the pathways, which are regulated by the Wnt pathway. This review focuses on the totality of Wnt target genes, the Wnt targetome, which is the clue to understand the manifold roles of the Wnt pathway within the cellular context.
Multidrug resistance (MDR) is a major limiting factor in the development and application of drug candidates. MDR caused by MRP-1 is known to be modulated by the nonsteroidal antiinflammatory drug ...indomethacin. We have synthesized and biologically evaluated a library of indomethacin analogues. The indomethacin-derived compound library was synthesized employing the Fischer−indole synthesis as the key transformation and making use of a “resin-capture-release” strategy. Sixty representative members of the library were evaluated in a cell biological cytotoxicity assay employing the MRP-1 expressing human glioblastoma cell line T98G as a model system. Nine of the 60 tested derivatives increased the doxorubicin-mediated cytotoxicity at a comparable or higher level than indomethacin itself. Analysis of these derivatives revealed an interesting structure−function relationship. Most remarkably, two substances increased the toxicity, when doxorubicin was used at clinically relevant low concentrations, at a higher degree than indomethacin.
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