Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle

Retinoid metabolism and all-trans retinoic acid-induced growth inhibition in head and neck squamous cell carcinoma cell lines.

Retinoids can reverse potentially premalignant lesions and prevent second primary tumours in patients with head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been reported that acquired resistance to all-trans retinoic acid (RA) in leukaemia is associated with decreased plasma peak levels, probably the result of enhanced retinoid metabolism. The aim of this study was to investigate the metabolism of retinoids and relate this to growth inhibition in HNSCC. Three HNSCC cell lines were selected on the basis of a large variation in the all-trans RA-induced growth inhibition. Cells were exposed to 9.5 nM (radioactive) for 4 and 24 h, and to 1 and 10 microM (nonradioactive) all-trans RA for 4, 24, 48 and 72 h, and medium and cells were analysed for retinoid metabolites. At all concentrations studied, the amount of growth inhibition was proportional to the extent at which all-trans-, 13- and 9-cis RA disappeared from the medium as well as from the cells. This turnover process coincided with the formation of a group of as yet unidentified polar retinoid metabolites. The level of mRNA of cellular RA-binding protein II (CRABP-II), involved in retinoid homeostasis, was inversely proportional to growth inhibition. These findings indicate that for HNSCC retinoid metabolism may be associated with growth inhibition

Regulation of stem cell differentiation by histone methyltransferases and demethylases

The generation of different cell types from stem cells containing identical genetic information and their organization into tissues and organs during development is a highly complex process that requires defined transcriptional programs. Maintenance of such programs is epigenetically regulated and the factors involved in these processes are often essential for development. The activities required for cell-fate decisions are frequently deregulated in human tumors, and the elucidation of the molecular mechanisms that regulate these processes is therefore important for understanding both developmental processes and tumorigenesis

PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation

Poly ADP-ribose polymerase (PARP) is a protein involved in single strand break repair. Recently, PARP inhibitors have shown considerable promise in the treatment of several cancers, both in monotherapy and in combination with cytotoxic agents. Synthetic lethal action of PARP inhibitors has been observed in tumors with mutations in double strand break repair pathways. In addition, PARP inhibition potentially enhances sensitivity of tumor cells to DNA damaging agents, including radiotherapy. Aim of this study is to determine the radiosensitizing properties of the PARP inhibitor Olaparib in childhood medulloblastoma, ependymoma and high grade glioma (HGG). Increased PARP1 expression was observed in medulloblastoma, ependymoma and HGG, as compared to non-neoplastic brain tissue. Pediatric high grade glioma, medulloblastoma and ependymoma gene expression profiling revealed that high PARP1 expression is associated with poor prognosis. Cell growth inhibition assays with Olaparib resulted in differential sensitivity, with IC50 values ranging from 1.4 to 8.4 μM, irrespective of tumor type and PARP1 protein expression. Sensitization to radiation was observed in medulloblastoma, ependymoma and HGG cell lines with subcytotoxic concentrations of Olaparib, which coincided with persistence of double strand breaks. Combining PARP inhibitors with radiotherapy in clinical studies in childhood high grade brain tumors may improve therapeutic outcome

Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1¿ strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression

MicroRNA-106b~25 cluster is upregulated in relapsed MLL-rearranged pediatric acute myeloid leukemia

The most important reason for therapy failure in pediatric acute myeloid leukemia (AML) is relapse. In order to identify miRNAs that contribute to the clonal evolution towards relapse in pediatric AML, miRNA expression profiling of 127 de novo pediatric AML cases were used. In the diagnostic phase, no miRNA signatures could be identified that were predictive for relapse occurrence, in a large pediatric cohort, nor in a nested mixed lineage leukemia (MLL)-rearranged pediatric cohort. AML with MLL- rearrangements are found in 15-20% of all pediatric AML samples, and reveal a relapse rate up to 50% for certain translocation partner subgroups. Therefore, microRNA expression profiling of six paired initial diagnosis-relapse MLL-rearranged pediatric AML samples (test cohort) and additional eight paired initial diagnosisrelapse samples with MLL-rearrangements (validation cohort) was performed. A list of 53 differentially expressed miRNAs was identified of which the miR-106b~25 cluster, located in intron 13 of MCM7, was the most prominent. These differentially expressed miRNAs however could not predict a relapse in de novo AML samples with MLLrearrangements at diagnosis. Furthermore, higher mRNA expression of both MCM7 and its upstream regulator E2F1 was found in relapse samples with MLL-rearrangements. In conclusion, we identified the miR-106b~25 cluster to be upregulated in relapse pediatric AML with MLL-rearrangements

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds

Background: Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. Methods: Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. Results: Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. Conclusions: This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors

Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (Rpearson  > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSChigh (>0·03% LSC) from LSClow (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification