MYCN amplification plus 1p36 loss of heterozygosity predicts ultra high risk in bone marrow metastatic neuroblastoma.

BACKGROUND: This study aimed to better understand the prognostic effect of multiple genetic markers and identify more subpopulations at ultra high risk of poor outcome in bone marrow (BM) metastatic neuroblastoma (NB). METHODS: We screened the MYCN, 1p36 and 11q23 loss of heterozygosity (LOH) statuses of 154 patients by interphase fluorescence in situ hybridization of BM cells. The clinical characteristics of patients with the three markers and their associations with prognosis were analysed. RESULTS: MYCN amplification and LOH at 1p36 and 11q23 were identified in 16.2%, 33.1% and 30.5% of patients, respectively. There were strong associations between MYCN amplification and 1p36 LOH as well as 11q23 LOH. Both MYCN amplification and 1p36 LOH were strongly associated with high levels of lactate dehydrogenase (LDH) and neuron-specific enolase, more than 3 metastatic organs, and more events. 11q23 LOH occurred mainly in patients older than 18 months, and those who had high LDH levels. In univariate analysis, patients with MYCN amplification had poorer prognosis than those without. Patients with 1p36 LOH had a 3-year event-free survival (EFS) and overall survival lower than those without. 11q23 LOH was associated with poorer EFS only for patients without MYCN amplification. In a multivariate model, MYCN amplification was independently associated with decreased EFS in all cohorts. 11q23 LOH was an independent prognostic factor for patients without MYCN amplification, whereas 1p36 LOH was not an independent marker regardless of MYCN amplification. Compared with all cohorts, patients with both MYCN amplification and 1p36 LOH had the worst outcome and clinical features. CONCLUSIONS: Patients with both MYCN amplification and 1p36LOH had the worst survival rate, indicating an ultra high-risk group. Our results may be applied in clinical practice for accurate risk stratification in future studies.

Downregulating CREBBP inhibits proliferation and cell cycle progression and induces daunorubicin resistance in leukemia cells.

Low expression levels of CREB­binding protein (CREBBP) have been demonstrated to be associated with high minimal residual disease at the end of induction therapy and adverse long­term outcomes in pediatric patients with acute lymphoblastic leukemia (ALL). However, the effect of low CREBBP expression on the prognosis of ALL has not yet been investigated. In the present study, CREBBP was downregulated and overexpressed in ALL cell lines (Jurkat and Reh). Sensitivity to chemotherapy and cell proliferation activity was determined via a Cell Counting Kit­8 assay. Cell cycle analysis was performed using flow cytometry. Immunofluorescence confocal microscopy and co­immunoprecipitation (Co­IP) assays were performed to determine the interaction between CREBBP and E2F transcription factor 3a (E2F3a). The binding of CREBBP to downstream gene caspase 8 associated protein 2 (CASP8AP2) promoters was assessed using a chromatin immunoprecipitation assay, and mRNA expression levels were detected via reverse transcription­quantitative PCR. Western blot analysis was performed to detect protein expression of CREBBP, E2F3a and CASP8AP2. Downregulation of CREBBP increased the IC50 value of daunorubicin; however, no significant affects were observed on the IC50 values of vincristine and L­asparaginase. Furthermore, downregulation of CREBBP notably inhibited leukemia cell proliferation, accumulated cells in the G0/G1 phase and decreased cell proportions in the S and G2/M phases. Co­IP analysis demonstrated that CREBBP interacted with E2F3a, a transcription factor involved in G1/S transition. Immunofluorescence confocal microscopy indicated co­localization of CREBBP and E2F3a at the cell nucleus. Furthermore, E2F3a protein expression decreased in CREBBP RNA interference treated Jurkat and Reh cells. CASP8AP2, a target gene of E2F3a, was also identified to be a downstream gene of CREBBP. In addition, decreased IC50 value and cell proportions in the G0/G1 phase, accelerated cell proliferation and upregulated E2F3a and CASP8AP2 expression were exhibited in CREBBP overexpressed cells. Taken together, the results of the present study suggested that CREBBP downregulation affects proliferation and cell cycle progression in leukemia cells, potentially via the interaction and regulation of E2F3a, resulting in chemotherapy resistance. Thus, targeting CREBBP may be a therapeutic strategy for treating pediatric patients with ALL.

Chromosome band 11q23 deletion predicts poor prognosis in bone marrow metastatic neuroblastoma patients without MYCN amplification.

BACKGROUND: Interphase fluorescence in situ hybridization (FISH) of bone marrow cells has been confirmed to be a direct and valid method to assess the v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification in patients with bone marrow metastatic neuroblastoma. MYCN amplification alone, however, is insufficient for pretreatment risk stratification. Chromosome band 11q23 deletion has recently been included in the risk stratification of neuroblastoma. In the present study, we aimed to evaluate the biological characteristics and prognostic impact of 11q23 deletion and MYCN amplification in patients with bone marrow metastatic neuroblastoma. METHODS: We analyzed the MYCN and 11q23 statuses of 101 patients with bone marrow metastatic neuroblastoma using interphase FISH of bone marrow cells. We specifically compared the biological characteristics and prognostic impact of both aberrations. RESULTS: MYCN amplification and 11q23 deletion were seen in 12 (11.9%) and 40 (39.6%) patients. The two markers were mutually exclusive. MYCN amplification occurred mainly in patients with high lactate dehydrogenase (LDH) and high neuron-specific enolase (NSE) levels (both P < 0.001), and MYCN-amplified patients had more events (tumor relapse, progression, or death) than MYCN-normal patients (P = 0.004). 11q23 deletion was associated only with age (P = 0.001). Patients with MYCN amplification had poorer outcomes than those with normal MYCN (3-year event-free survival [EFS] rate: 8.3 ± 8.0% vs. 43.8 ± 8.5%, P < 0.001; 3-year overall survival [OS] rate: 10.4 ± 9.7% vs. 63.5% ± 5.7%, P < 0.001). 11q23 deletion reflected a poor prognosis only for patients with normal MYCN (3-year EFS rate: 34.3 ± 9.5% vs. 53.4 ± 10.3%, P = 0.037; 3-year OS rate: 42.9 ± 10.4% vs. 75.9 ± 6.1%, P = 0.048). Those with both MYCN amplification and 11q23 deletion had the worst outcome (P < 0.001). CONCLUSIONS: Chromosome band 11q23 deletion predicts poor prognosis only in bone marrow metastatic neuroblastoma patients without MYCN amplification. Combined assessment of the two markers was much superior to single-marker assessment in recognizing the patients at a high risk of disease progression.

The prognostic potential of coilin in association with p27 expression in pediatric acute lymphoblastic leukemia for disease relapse.

BACKGROUND: Cajal body (CB) is a nucleic organelle where small nuclear ribonucleoproteins undergo modification, maturation, splicing and/or assembly. Coilin is the marker structural protein of CBs. The expression level and cellular localization of coilin is sensitive to chemotherapeutic reagents, such as cisplatin. The gene of cyclin-dependent kinase inhibitor 1B (p27) is located with a high incidence translocation region of leukemic chromosomes, and its expression was of prognosis values in a variety of adult leukemia types. The exact profile and associated functions of coilin, as well as p27, in children's acute lymphoblastic leukemia (ALL) is obscure. METHODS: Bone marrow samples from 144 patients with ALL were collected. The expression levels of coilin and p27 were detected by qRT-PCR. The patient cohort was divided into low and high groups of coilin and p27 respectively. The prognosis and clinicobiological characteristics of different groups were investigated, especially focused on the treatment outcome. Leukemia cells of Reh or RS4;11 were exposed to different concentrations of DNR, prior to the detection for morphological changes of coilin by immunofluorescence. In Reh cells, lentivirus sh-coilin was used to silence coilin expression. Western blotting was used to detect coilin and p27 expression; flow cytometry was used for cell cycle and apoptosis assay; MTS method was used for measuring cell viability to examine the drug sensitivity of DNR. RESULTS: In this study, we found that daunorubicin was able to induce significant morphological changes of CBs in Reh and RS4;11 cells. Knockdown the expression of coilin increased the sensitivity to daunorubicin and inhibited the expression of p27 in Reh cells, and led to increased apoptosis. Importantly, not only the levels of coilin and p27 mRNA expression at initial diagnosis ALL children are markedly higher than those at complete remission (CR), but also both coilin and p27 expression in the relapsed patients was observed significantly higher comparing to the continuous CR patients. The 4-year EFS and RFS indicated that low levels of both coilin and p27 group favored better prognosis (p < 0.05). CONCLUSIONS: Our results indicated that consideration of coilin and p27 levels could be a prognostic reference for predicting the outcome of pediatric ALL patients, especially for disease recurrence. Reduction of coilin expression was sufficient to increase the sensitivity of leukemic cells to daunorubicin treatments, and during which possibly involved functions of p27 in cell cycle regulation and its effects on cell apoptosis.

[Post-transcriptional regulation of chicken PPARγ transcript variant 3 by upstream open reading frame].

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript variants contain upstream open reading frames (uORFs) in their 5'untranslated region (5'TR). To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγ gene, we constructed wild-type (psiCHECK2-cPPARγ3-5'UTR-WT) and a uORF mutant (the upstream ATG (uATG) was mutated to stop codon TGA) 5'UTR reporters (psiCHECK2-cPPARγ3- 5'UTR-Mut) of chicken PPARγ transcript variant 3 (cPPARγ3). These two reporters were individually transfected into immortalized chicken pre-adipocytes (ICPA) and DF1 cells, and the renilla luciferase (hRluc) activity and mRNA expression level were detected by reporter assay and qRT-PCR. The results showed that the hRluc activity of the mutated 5'UTR was significantly higher than that of the wild-type 5'UTR in ICPA cells (P<0.01), and the hRluc activity of the mutated 5'UTR tended to be higher than that of the wild-type 5'UTR in DF1 cells, but this difference did not reach statistical significance (P>0.05). The qRT-PCR analysis showed, in ICPA cells, the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct (P<0.01). In DF1 cells, the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct, but this difference did not reach statistical significance (P>0.05). To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF, we constructed the expression plasmids bearing the full-length coding region of chicken PPARγ gene plus either wild-type or mutant uORF 5'UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5'UTR of the cPPARγ3 inhibits its translation.

Genetic and epigenetic regulation of PPARγ during adipogenesis.

Peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and adipose tissue development. It also plays crucial roles in many other biological processes, including lipid and glucose metabolism and energy homeostasis. Recently, evidence has been accumulating that the PPARγ gene is not only genetically regulated, but also epigenetically regulated by DNA methylation, histone modification, non-coding RNA and chromosome remodeling. In this review, we summarize the advances in the genetic and epigenetic regulation of the PPARγ gene during adipogenesis, and discuss future research directions and trends for the study of its regulation.

MYCN amplification predicts poor prognosis based on interphase fluorescence in situ hybridization analysis of bone marrow cells in bone marrow metastases of neuroblastoma.

BACKGROUND: MYCN gene amplification is related to risk stratification. Therefore it is important to identify accurately the level of the MYCN gene as early as possible in neuroblastoma (NB); however, for patients with bone marrow (BM) metastasis who need chemotherapy before surgery, timely detection of the MYCN gene is not possible due to the unavailability of primary tumors. METHODS: MYCN gene status was evaluated in 81 BM metastases of NB by interphase fluorescence in situ hybridization (FISH) analysis of BM cells. The clinicobiological characteristics and prognostic impact of MYCN amplification in NB metastatic to BM were analyzed. RESULTS: MYCN amplification was found in 16% of patients with metastases, and the results were consistent with the primary tumors detected by pathological tissue FISH. MYCN amplification was associated with age, lactate dehydrogenase (LDH) levels and prognosis (P = 0.038, P < 0.001, P = 0.026). Clinical outcome was poorer in patients with MYCN amplification than in those without amplification (3-year EFS 28.8 ± 13.1 vs. 69.7 ± 5.7%, P = 0.005; 3-year OS 41.5 ± 14.7 vs. 76.7 ± 5.5%, P = 0.005). CONCLUSIONS: MYCN amplification predicts a poor outcome in NB metastatic to BM, and interphase FISH of bone marrow cells provides a timely direct and valid method to evaluate the MYCN gene status.

Functional analysis of the upstream regulatory region of chicken miR-17-92 cluster.

miR-17-92 cluster plays important roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis. The transcriptional regulation of miR-17-92 cluster has been extensively studied in mammals, but not in birds. To date, avian miR-17-92 cluster genomic structure has not been fully determined. The promoter location and sequence of miR-17-92 cluster have not been determined, due to the existence of a genomic gap sequence upstream of miR-17-92 cluster in all the birds whose genomes have been sequenced. In this study, genome walking was used to close the genomic gap upstream of chicken miR-17-92 cluster. In addition, bioinformatics analysis, reporter gene assay and truncation mutagenesis were used to investigate functional role of the genomic gap sequence. Genome walking analysis showed that the gap region was 1704 bp long, and its GC content was 80.11%. Bioinformatics analysis showed that in the gap region, there was a 200 bp conserved sequence among the tested 10 species (Gallus gallus, Homo sapiens, Pan troglodytes, Bos taurus, Sus scrofa, Rattus norvegicus, Mus musculus, Possum, Danio rerio, Rana nigromaculata), which is core promoter region of mammalian miR-17-92 host gene (MIR17HG). Promoter luciferase reporter gene vector of the gap region was constructed and reporter assay was performed. The result showed that the promoter activity of pGL3-cMIR17HG (-4228/-2506) was 417 times than that of negative control (empty pGL3 basic vector), suggesting that chicken miR-17-92 cluster promoter exists in the gap region. To further gain insight into the promoter structure, two different truncations for the cloned gap sequence were generated by PCR. One had a truncation of 448 bp at the 5'-end and the other had a truncation of 894 bp at the 3'-end. Further reporter analysis showed that compared with the promoter activity of pGL3-cMIR17HG (-4228/-2506), the reporter activities of the 5'-end truncation and the 3'-end truncation were reduced by 19.82% and 60.14%, respectively. These data demonstrated that the important promoter region of chicken miR-17-92 cluster is located in the -3400/-2506 bp region. Our results lay the foundation for revealing the transcriptional regulatory mechanisms of chicken miR-17-92 cluster.

MicroRNA-24 modulates aflatoxin B1-related hepatocellular carcinoma prognosis and tumorigenesis.

MicroRNA-24 (miR-24) may be involved in neoplastic process; however, the role of this microRNA in the hepatocellular carcinoma (HCC) related to aflatoxin B1 (AFB1) has not been well elaborated. Here, we tested miR-24 expression in 207 pathology-diagnosed HCC cases from high AFB1 exposure areas and HCC cells. We found that miR-24 was upregulated in HCC tumor tissues relative to adjacent noncancerous tissue samples, and that the high expression of miR-24 was significantly correlated with larger tumor size, higher microvessel density, and tumor dedifferentiation. Additionally, this microRNA overexpression modified the recurrence-free survival (relative hazard ratio [HR], 4.75; 95% confidence interval [CI], 2.66-8.47) and overall survival (HR = 3.58, 95% CI = 2.34-5.46) of HCC patients. Furthermore, we observed some evidence of joint effects between miR-24 and AFB1 exposure on HCC prognosis. Functionally, miR-24 overexpression progressed tumor cells proliferation, inhibited cell apoptosis, and developed the formation of AFB1-DNA adducts. These results indicate for the first time that miR-24 may modify AFB1-related HCC prognosis and tumorigenesis.

HDAC inhibitor sodium butyrate augments the MEF2C enhancement of Nampt expression under hypoxia.

Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme for the salvage biosynthesis of nicotinamide adenine dinucleotide (NAD). Although elevated level of Nampt expression has been observed in various cancers, the involvement of Nampt promoter regulation was not well understood. We have identified a cluster of MEF2 recognition sites upstream of the functional hypoxia response elements (HREs) within the human Nampt promoter, and demonstrated that the two MEF2 sites at -1272 and -1200 were functional to upregulate the promoter activity by luciferase reporter assays. The Nampt promoter was able to be activated cooperatively following hypoxic stimulation by CoCl2 treatment with associated MEF2C overexpression. During the investigation on MEF2C regulation of endogenous Nampt expression in HeLa cells, the most significant enhancement of Nampt expression observed was by overexpression of MEF2C in combination with sodium butyrate exposure. By chromatin immunoprecipitation with a MEF2C anti-body, we found that MEF2C indeed interacted with endogenous Nampt promoter. The requirement of HDAC inhibition for the MEF2C enhancement of Nampt transcription was verified by RNAi of HDAC. Our results were in support of reports indicating that MEF2 family transcription factors interacted with HDACs and regulated downstream gene expression at the epigenetic levels. Our study provided important evidence to demonstrate the sophisticated mechanism of endogenous Nampt promoter regulation, and therefore, will help to better understand the Nampt overexpression in cancer progression, especially in the context of MEF2C upregulation which frequently occurred in cancer development and drug resistance.

HIF1 regulates WSB-1 expression to promote hypoxia-induced chemoresistance in hepatocellular carcinoma cells.

WSB-1 is involved in DNA damage response by targeting homeodomain-interacting protein kinase 2 (HIPK2) for ubiquitination and degradation. Here, we report that hypoxia significantly up-regulates the expression of WSB-1 in human hepatocellular carcinoma (HCC) cells. We also provide evidence that WSB-1 is a target of hypoxia-inducible factor 1 (HIF-1). Silencing the expression of HIF-1α in HCC cells by RNA interference abolishes hypoxia-induced WSB-1 expression. Using chromatin immunoprecipitation and luciferase reporter assays, we identified a HRE of the WSB-1 gene. Moreover, silencing the expression of WSB-1 by RNA interference rescues HIPK2 expression in hypoxic HCC cells and promotes etoposide-induced cell death in hypoxic HCC cells. Taken together, these data shed light on the mechanisms underlying hypoxia-induced chemoresistance in HCC cells.

[Recent advance in DNA epigenetic modifications-the sixth base in the genome].

Recently, 5-hydroxymethyl cytosine (5-hmC) has been discovered as a naturally existed component of normal mammalian genomic DNAs, and it is generally accepted as the sixth base in the genome. This review will introduce the recent advances in the researches on 5-hmC of its formation, tissue-specific distribution, the roles in cell differentiation and gene expression regulation, and the connections as a epigenetic marker with diseases, such as various cancers. We also summarized the current development of the methodologies to detect methylated or hydroxymetholated cytosines of cells at the genomic levels.