In renal cell carcinoma the PTEN splice variant PTEN-Δ shows similar function as the tumor suppressor PTEN itself

BACKGROUND: Loss of PTEN is involved in tumor progression of several tumor entities including renal cell carcinoma (RCC). During the translation process PTEN generates a number of splice variants, including PTEN-Δ. We analyzed the impact of PTEN-Δ in RCC progression. METHODS: In specimens of RCC patients the expression of PTEN-Δ and PTEN was quantified. The PTEN expressing RCC cell line A498 and the PTEN deficient 786-O cell line were stably transfected with the PTEN-Δ or PTEN transcript. In Caki-1 cells that highly express PTEN-Δ, this isoform was knocked down by siRNA. Cell migration, adhesion, apoptosis and signaling pathways activities were consequently analyzed in vitro. RESULTS: Patients with a higher PTEN-Δ expression had a longer lymph node metastasis free and overall survival. In RCC specimens, the PTEN-Δ expression correlated with the PTEN expression. PTEN-Δ as well as PTEN induced a reduced migration when using extracellular matrix (ECM) compounds as chemotaxins. This effect was confirmed by knockdown of PTEN-Δ, inducing an enhanced migration. Likewise a decreased adhesion on these ECM components could be shown in PTEN-Δ and PTEN transfected cells. The apoptosis rate was slightly increased by PTEN-Δ. In a phospho-kinase array and Western blot analyses a consequently reduced activity of AKT, p38 and JNK could be shown. CONCLUSIONS: We could show that the PTEN splice variant PTEN-Δ acts similar to PTEN in a tumor suppressive manner, suggesting synergistic effects of the two isoforms. The impact of PTEN-Δ in context of tumor progression should thus be taken into account when generating new therapeutic options targeting PTEN signaling in RCC

Genomisches Imprinting Beckwith-Wiedemann-Syndrom assoziierter Gene

In der vorliegenden Arbeit wurde das Imprinting von Genen der Chromosomenregion 11p15.5 des Menschen und des orthologen murinen Abschnitts 7F5 untersucht. Bei der Analyse der humanen Gene H19, IGF2 und KCNQ1OT1 stand deren Regulation durch differentiell methylierte Regionen (DMR) und die Identifizierung von Methylierungsfehlern bei Patienten mit Verdacht auf Beckwith-Wiedemann Syndrom (BWS) im Vordergrund. Hierzu wurden unmethylierte Cytosinnukleotide durch Bisulfitbehandlung in Uracilnukleotide umgewandelt und PCR-amplifizierte DNA-Fragmente sequenziert. Die elterliche Herkunft der Allele wurde mit Hilfe von Einzelnukleotidpolymorphismen (SNP) bestimmt. Während in der H19-Promotorregion in Lymphozyten eine nur tendenziell allelspezifische Methylierung festgestellt werden konnte, wurde im B1-Repeat der H19/IGF2-Region in allen Kontroll- und 20 Patienten-DNAs eine spezifische Methylierung des väterlichen Allels nachgewiesen. Vier BWS-DNAs zeigten hingegen eine nahezu vollständige Hypomethylierung. In der zweIn the presented study genomic imprinting of genes located on human chromosome 11p15.5 and the murine orthologous region 7F5 was analysed. Human genes H19, IGF2 and KCNQ1OT1 were examined focussing on their regulation by differentially methylated regions (DMR) and the identification of methylation aberrations in cases with Beckwith-Wiedemann syndrome. Unmethylated cytosines were transformed into uraciles by treatment with sodium bisulfite followed by sequencing of the PCR-amplified DNA fragments. Parental origin of the alleles was determined using single nucleotide polymorphisms (SNP). While analysis of the H19 promoter detected only a faint bias for allelespecific methylation in lymphocytes, for the B1-repeat of the H19/IGF2-region exclusive methylation of the paternal allele was detected in all control- and 20 BWS-DNAs. Four BWS cases showed a nearly complete lack of methylated cytosines (hypomethylation) in B1 on both parental alleles. In the second DMR associated with BWS (KvDMR) allelespecific methylat

Additional file 3: of In renal cell carcinoma the PTEN splice variant PTEN-Δ shows similar function as the tumor suppressor PTEN itself

Figure S3. Human phospho-kinase array (Roche) of transfected 786-O and A498 cells. Protein extracts were obtained from PTEN-Δ and PTEN transfected cells and analyzed concerning the phosphorylation status of 46 intracellular signaling kinases. The activity of the kinases AKT, JNK and p38 are highlighted with red boxes. (PDF 111 kb

Kaiso mediates human ICR1 methylation maintenance and H19 transcriptional fine regulation

Background Genomic imprinting evolved in a common ancestor to marsupials and eutherian mammals and ensured the transcription of developmentally important genes from defined parental alleles. The regulation of imprinted genes is often mediated by differentially methylated imprinting control regions (ICRs) that are bound by different proteins in an allele-specific manner, thus forming unique chromatin loops regulating enhancer-promoter interactions. Factors that maintain the allele-specific methylation therefore are essential for the proper transcriptional regulation of imprinted genes. Binding of CCCTC-binding factor (CTCF) to the IGF2/H19-ICR1 is thought to be the key regulator of maternal ICR1 function. Disturbances of the allele-specific CTCF binding are causative for imprinting disorders like the Silver-Russell syndrome (SRS) or the Beckwith-Wiedemann syndrome (BWS), the latter one being associated with a dramatically increased risk to develop nephroblastomas. Methods Kaiso binding to the human ICR1 was detected and analyzed by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA). The role of Kaiso-ICR1 binding on DNA methylation was tested by lentiviral Kaiso knockdown and CRISPR/Cas9 mediated editing of a Kaiso binding site. Results We find that another protein, Kaiso (ZBTB33), characterized as binding to methylated CpG repeats as well as to unmethylated consensus sequences, specifically binds to the human ICR1 and its unmethylated Kaiso binding site (KBS) within the ICR1. Depletion of Kaiso transcription as well as deletion of the ICR1-KBS by CRISPR/Cas9 genome editing results in reduced methylation of the paternal ICR1. Additionally, Kaiso affects transcription of the lncRNA H19 and specifies a role for ICR1 in the transcriptional regulation of this imprinted gene. Conclusions Kaiso binding to unmethylated KBS in the human ICR1 is necessary for ICR1 methylation maintenance and affects transcription rates of the lncRNA H19

Additional file 2: of In renal cell carcinoma the PTEN splice variant PTEN-Δ shows similar function as the tumor suppressor PTEN itself

Figure S2. Influence of PTEN-∆ and PTEN on proliferation. Proliferation was determined by BrdU incorporation. Differences are shown as percentage of the transfection control cells (pcDNA3 transfected cells). (PDF 90 kb

Additional file 1: of In renal cell carcinoma the PTEN splice variant PTEN-Δ shows similar function as the tumor suppressor PTEN itself

Figure S1. Western blot of A498 and 786-O cells. Protein extracts of A498 and 786-O cells were analyzed concerning the expression value of PTEN. (PDF 89 kb

Additional file 2: Table S1. of Kaiso mediates human ICR1 methylation maintenance and H19 transcriptional fine regulation

Influence of Kaiso on other ICRs. Other ICRs than the ICR1 based on (Court et al. [43]) on different chromosomes were analysed for the presence of KBS and their number listed. In addition, the presence of ZPF57 target sites (ZFP57TS) was analysed (Y = yes, N = no) as well as the Kaiso knockdown effect on the methylation of the DMR and the transcription of flanking imprinted genes of the respective ICRs (n.d. = not determined, NO = no change observed, UP = upregulated, DOWN = downregulated). (XLSX 13 kb