Genes Controlling Cell Proliferation Research Articles

DeSUMOylation of chromatin-bound proteins limits the rapid transcriptional reprogramming induced by daunorubicin in acute myeloid leukemias.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces rapid (3h) and broad transcriptional changes in AML cells. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, in particular at active promoters and enhancers. Surprisingly, inhibition of SUMOylation with ML-792 (SUMO E1 inhibitor), dampens DNR-induced transcriptional reprogramming. Quantitative proteomics shows that the proteins deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. DNR leads to a reconfiguration of chromatin loops engaging CTCF- and SUMO-bound NFKB2 promoter with a distal cis-regulatory region and inhibition of SUMOylation with ML-792 prevents these changes.

Transcriptome analysis reveals key transcription factors and pathways of polian vesicle associated with cell proliferation in Vibrio splendidus-challenged Apostichopus japonicus

Polian vesicle is thought to produce coelomocytes and contribute to the sea cucumber's immune system. Our previous work has indicated that polian vesicle was responsible for cell proliferation at 72 h post pathogenic challenge. However, the transcription factors related to the activation of effector factors and the molecular process behind this remained unknown. In this study, to reveal the early functions of polian vesicle in response to the microbe, a comparative transcriptome sequencing of polian vesicle in V. splendidus-challenged Apostichopus japonicus, including normal group (PV 0 h), pathogen challenging for 6 h (PV 6 h) and 12 h (PV 12 h) was performed. Compared PV 0 h to PV 6 h, PV 0 h to PV 12 h, and PV 6 h to PV 12 h, we found 69, 211, and 175 differentially expressed genes (DEGs), respectively. KEGG enrichment analysis revealed the DEGs, including several transcription factors such as fos, FOS-FOX, ATF2, egr1, KLF2, and Notch3 between PV 6 h and PV 12 h were consistently enriched in MAPK, Apelin and Notch3 signaling pathways related to cell proliferation compared with that in PV 0 h. Important DEGs involved in cell growth were chosen, and their expression patterns were almost the same as the transcriptome profile analysis by qPCR. Protein interaction network analysis indicated that two DEGs of fos and egr1 were probably significant as key candidate genes controlling cell proliferation and differentiation in polian vesicle after pathogenic infection in A. japonicus. Overall, our analysis demonstrates that polian vesicles may play an essential role in regulating proliferation via transcription factors-mediated signaling pathway in A. japonicus and provide new insights into hematopoietic modulation of polian vesicles in response to pathogen infection.

The Four Homeostasis Knights: In Balance upon Post-Translational Modifications.

A cancer outcome is a multifactorial event that comes from both exogenous injuries and an endogenous predisposing background. The healthy state is guaranteed by the fine-tuning of genes controlling cell proliferation, differentiation, and development, whose alteration induces cellular behavioral changes finally leading to cancer. The function of proteins in cells and tissues is controlled at both the transcriptional and translational level, and the mechanism allowing them to carry out their functions is not only a matter of level. A major challenge to the cell is to guarantee that proteins are made, folded, assembled and delivered to function properly, like and even more than other proteins when referring to oncogenes and onco-suppressors products. Over genetic, epigenetic, transcriptional, and translational control, protein synthesis depends on additional steps of regulation. Post-translational modifications are reversible and dynamic processes that allow the cell to rapidly modulate protein amounts and function. Among them, ubiquitination and ubiquitin-like modifications modulate the stability and control the activity of most of the proteins that manage cell cycle, immune responses, apoptosis, and senescence. The crosstalk between ubiquitination and ubiquitin-like modifications and post-translational modifications is a keystone to quickly update the activation state of many proteins responsible for the orchestration of cell metabolism. In this light, the correct activity of post-translational machinery is essential to prevent the development of cancer. Here we summarize the main post-translational modifications engaged in controlling the activity of the principal oncogenes and tumor suppressors genes involved in the development of most human cancers.

DIPG-57. TRANSCRIPTOMIC AND PROTEOMIC ANALYSES OF DIPG RESPONSE TO ONC201

Diffuse Intrinsic Pontine Glioma (DIPG) is an incurable pediatric brain tumor. Current standard of care has shown no improvements in survival. Here, we report our study of ONC201, a first-in-class small molecule developed by Oncoceutics, Inc., against a panel of DIPG cells in vitro and in mouse orthotopic models. ONC201 inhibits signaling through dopamine receptor D2 (DRD2), a G protein-coupled receptor (GPCR). MTT assays revealed a delayed but more robust response to ONC201, as measured by IC50 values, in DIPGs with histone H3.3-K27M expression compared to cells expressing wildtype (WT) or K27M mutant histone H3.1. Interestingly, transcriptomic profiling identified an association of this response delay with an elevation of genes controlling the cellular unfolded protein response, lysosomal and vacuole organization, and a decline in nucleic acid biosynthetic genes. These cells were also more committed to neuronal and oligodendrocytic lineage specification. By contrast, WT-H3 DIPGs that survived ONC201 treatment were stem-like and exhibited altered expression of genes controlling cell proliferation and apoptosis induction, respectively. Single cell proteomics validated the increase in anti-apoptotic proteins in these cells. Intraperitoneal administration of ONC201 for 7-weeks in mice bearing pontine xenografts of histone H3.1-K27M mutant DIPGs, caused a complete blockade of tumor growth relative to untreated controls. However, identical treatment of animals with forebrain tumors resulted only in a partial reduction in tumor burden, suggesting that the tumor microenvironment may be involved in the differential effect. These data indicate that tumor intrinsic and extrinsic factors may contribute to the response of DIPG tumors to ONC201.

The S-phase-induced lncRNA SUNO1 promotes cell proliferation by controlling YAP1/Hippo signaling pathway.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Potent Anti-Cancer Properties of Phthalimide-Based Curcumin Derivatives on Prostate Tumor Cells.

Metastatic castration-resistant prostate cancer is commonly treated with chemotherapy, whose effect is less than satisfactory. This raised the need for novel agents for the treatment of prostate cancer. In the present study, five phthalimide-based curcumin derivatives were synthesized and completely characterized to assess improved stability, pharmacodynamics, and radical scavenging ability. To investigate the potential application in anti-cancer therapy, the anti-proliferative activity of the synthesized molecules was determined on aggressive prostate tumor cells. We demonstrated that the K3F21 derivative has increased potency compared to curcumin, in terms of GI50, anti-proliferative and anti-migrating activities. K3F21 inhibits anchorage-dependent and -independent growth of prostate cancer cells by altering the expression of key genes controlling cell proliferation, such as Cylins D1, B1 and B2, and apoptosis, among which Puma, Noxa, and Bcl-2 family members. Finally, the anti-cancer activity of K3F21 was demonstrated by the analysis of cancer-associated PI3K/AKT, ERK, and p38 signaling pathways.

HAfTs are novel lncRNA transcripts from aflatoxin exposure.

The transcriptome can reveal insights into precancer biology. We recently conducted RNA-Seq analysis on liver RNA from male rats exposed to the carcinogen, aflatoxin B1 (AFB1), for 90 days prior to liver tumor onset. Among >1,000 differentially expressed transcripts, several novel, unannotated Cufflinks-assembled transcripts, or HAfTs (Hepatic Aflatoxin Transcripts) were found. We hypothesized PCR-cloning and RACE (rapid amplification of cDNA ends) could further HAfT identification. Sanger data was obtained for 6 transcripts by PCR and 16 transcripts by 5’- and 3’-RACE. BLAST alignments showed, with two exceptions, HAfT transcripts were lncRNAs, >200nt without apparent long open reading frames. Six rat HAfT transcripts were classified as ‘novel’ without RefSeq annotation. Sequence alignment and genomic synteny showed each rat lncRNA had a homologous locus in the mouse genome and over half had homologous loci in the human genome, including at least two loci (and possibly three others) that were previously unannotated. While HAfT functions are not yet clear, coregulatory roles may be possible from their adjacent orientation to known coding genes with altered expression that include 8 HAfT-gene pairs. For example, a unique rat HAfT, homologous to Pvt1, was adjacent to known genes controlling cell proliferation. Additionally, PCR and RACE Sanger sequencing showed many alternative splice variants and refinements of exon sequences compared to Cufflinks assembled transcripts and gene prediction algorithms. Presence of multiple splice variants and short tandem repeats found in some HAfTs may be consequential for secondary structure, transcriptional regulation, and function. In summary, we report novel, differentially expressed lncRNAs after exposure to the genotoxicant, AFB1, prior to neoplastic lesions. Complete cloning and sequencing of such transcripts could pave the way for a new set of sensitive and early prediction markers for chemical hepatocarcinogens.

Diabetic Ephrin-B2-Stimulated Peripheral Blood Mononuclear Cells Enhance Poststroke Recovery in Mice.

Clinical trials of cell therapy in stroke favor autologous cell transplantation. To date, feasibility studies have used bone marrow-derived mononuclear cells, but harvesting bone marrow cells is invasive thus complicating bedside treatment. We investigated the therapeutic potential of peripheral blood-derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+) (500,000 cells), injected intravenously 18–24 hours after induced cerebral ischemia in mice. Infarct volume, neurological deficit, neurogenesis, angiogenesis, and inflammation were investigated as were the potential mechanisms of PB-MNC+ cells in poststroke neurorepair. At D3, infarct volume was reduced by 60% and 49% compared to unstimulated PB-MNC and PBS-treated mice, respectively. Compared to PBS, injection of PB-MNC+ increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglia/macrophage cell density, and upregulated TGF-β expression. At D14, microvessel density was decreased and functional recovery was enhanced compared to PBS-treated mice, whereas plasma levels of BDNF, a major regulator of neuroplasticity, were increased in mice treated with PB-MNC+ compared to the other two groups. Cell transcriptional analysis showed that ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation, and angiogenesis, as confirmed by adhesion and Matrigel assays. Conclusions. This feasibility study suggests that PB-MNC+ transplantation poststroke could be a promising approach but warrants further investigation. If confirmed, this rapid, noninvasive bedside cell therapy strategy could be applied to stroke patients at the acute phase.

Stroke and the Cell Therapy Saga: Towards a Safe, Swift and Efficient Utilization of cells.

The first clinical trials of cell therapy in stroke were first published in the 2000s and consisted of neural stems cells transplanted via the intracerebral pathway. Since mesenchymal stem cells showed similar capacities to differentiate into neural cells and allowed autologous cell transplantation, they were then preferentially studied, including diabetes and hypertension. More recently, bone marrow derived mononuclear cells were successfully transplanted in stroke with no need of culture processing, and simple collection by density gradient centrifugation rendering them immediately ready for use. They improve post-stroke neurological deficit in rodents and clinical trials have shown the feasibility of intra-arterial or intravenous administration. The underlying mechanisms are not yet understood. We investigated the therapeutic potential of peripheral blood derived mononuclear cells (PB-MNC) harvested from diabetic patients and stimulated by ephrin-B2 (PB-MNC+). We showed that intravenously injected PB-MNC+ after cerebral ischemia reduced infarct volume at day 3, increased cell proliferation in the peri-infarct area and the subventricular zone, decreased microglial cell density, and upregulated TGF-β expression. At D14, microvessel density was increased and functional recovery enhanced, whereas plasma levels of BDNF were increased in treated mice. Ephrin-B2 induced phenotype switching of PB-MNC by upregulating genes controlling cell proliferation, inflammation and angiogenesis, as confirmed by adhesion and Matrigel assays. PB-MNC+ transplantation in stroke is a promising approach and should be investigated for the development of rapid, non-invasive bedside cell therapy strategies in stroke.(Presented at the 1944th Meeting, July 19, 2017).

Genome-Wide Analysis of core Promoter Region Cytosine-Phosphate-Guanine Islands Hypermethylation Profiles In Chronic Hepatitis B Virus Patients In South Africa

Purpose: Hepatitis B virus (HBV) infection causes epigenetic changes through DNA hypermethylation. HBV integrates into the human genome thereby facilitating viral persistence. Infected hepatocytes express DNA methyltransferases that hypermethylate and inactivate integrated HBV genome inserts. This defence mechanism will methylate adjacent host DNA; this may result in the methylation of tumour suppressor genes and cell-cycle regulators leading to hepatocyte injury and malignancy. The aim of this study was to investigate for the presence of genome-wide promoter region cytosine-phosphate-guanine islands hypermethylation in HBV infected patients. Materials and methods: Affymetrix Human Promoter 1.0R microarray chip was used for microarray analysis of DNA obtained from liver tissue of 17 HBV-infected patients, 2 normal liver histopathology controls, 7 hepatocellular carcinoma (HCC), 2 from liver tissue surrounding HCC lesions and 4 from patients with autoimmune hepatitis. Microarray data was analysed using Partek Genomic Suite focusing on the core promoter region within 100bp from transcription start site. Partek Pathway was used for biological pathway analysis. The discovered hypermethylation patterns were validated using bisulfite DNA sequencing in an additional set of 59 samples. Results: HBV infected patients had significant hypermethylation compared to other study groups. Genes controlling hepatic inflammation, tissue regeneration and carcinogenesis were significantly affected (P<0.01). Age over 40 years, cirrhosis, hepatic inflammation and HBV e antigen status correlated with hypermethylation (P<0.01). On bisulfite DNA sequencing, Cyclin D3 gene promoter was particularly highly methylated. This was associated with HBV e antigen positive status, high viral load and genotype D (P=0.01). Conclusions: HBV-induced epigenetic changes were widespread, affecting genes controlling cell growth, differentiation, proliferation and apoptosis. These changes may explain the development of hepatic inflammation, fibrosis and malignancy observed in patients with chronic HBV infection.

STATs: An Old Story, Yet Mesmerizing.

Signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors that have a key role in cell fate. STATs, a protein family comprised of seven members, are proteins which are latent cytoplasmic transcription factors that convey signals from the cell surface to the nucleus through activation by cytokines and growth factors. The signaling pathways have diverse biological functions that include roles in cell differentiation, proliferation, development, apoptosis, and inflammation which place them at the center of a very active area of research. In this review we explain Janus kinase (JAK)/STAT signaling and focus on STAT3, which is transient from cytoplasm to nucleus after phosphorylation. This procedure controls fundamental biological processes by regulating nuclear genes controlling cell proliferation, survival, and development. In some hematopoietic disorders and cancers, overexpression and activation of STAT3 result in high proliferation, suppression of cell differentiation and inhibition of cell maturation. This article focuses on STAT3 and its role in malignancy, in addition to the role of microRNAs (miRNAs) on STAT3 activation in certain cancers.

The Oncogenic Roles of DICER1 RNase IIIb Domain Mutations in Ovarian Sertoli-Leydig Cell Tumors

DICER1, an endoribonuclease required for microRNA (miRNA) biogenesis, is essential for embryogenesis and the development of many organs including ovaries. We have recently identified somatic hotspot mutations in RNase IIIb domain of DICER1 in half of ovarian Sertoli-Leydig cell tumors, a rare class of sex-cord stromal cell tumors in young women. These hotspot mutations lost IIIb cleavage activity of DICER1 in vitro and failed to produce 5p-derived miRNAs in mouse Dicer1-null ES cells. However, the oncogenic potential of these hotspot DICER1 mutations has not been studied. Here, we further revealed that the global expression of 5p-derived miRNAs was dramatically reduced in ovarian Sertoli-Leydig cell tumors carrying DICER1 hotspot mutations compared with those without DICER1 hotspot mutation. The miRNA production defect was associated with the deregulation of genes controlling cell proliferation and the cell fate. Using an immortalized human granulosa cell line, SVOG3e, we determined that the D1709N-DICER1 hotspot mutation failed to produce 5p-derived miRNAs, deregulated the expression of several genes that control gonadal differentiation and cell proliferation, and promoted cell growth. Re-expression of let-7 significantly inhibited the growth of D1709N-DICER1 SVOG3e cells, accompanied by the suppression of key regulators of cell cycle control and ovarian gonad differentiation. Taken together, our data revealed that DICER1 hotspot mutations cause systemic loss of 5p-miRNAs that can both drive pseudodifferentiation of testicular elements and cause oncogenic transformation in the ovary.

Small molecule inhibitors of IRES-mediated translation

Many genes controlling cell proliferation and survival (those most important to cancer biology) are now known to be regulated specifically at the translational (RNA to protein) level. The internal ribosome entry site (IRES) provides a mechanism by which the translational efficiency of an individual or group of mRNAs can be regulated independently of the global controls on general protein synthesis. IRES-mediated translation has been implicated as a significant contributor to the malignant phenotype and chemoresistance, however there has been no effective means by which to interfere with this specialized mode of protein synthesis. A cell-based empirical high-throughput screen was performed in attempt to identify compounds capable of selectively inhibiting translation mediated through the IGF1R IRES. Results obtained using the bicistronic reporter system demonstrate selective inhibition of second cistron translation (IRES-dependent). The lead compound and its structural analogs completely block de novo IGF1R protein synthesis in genetically-unmodified cells, confirming activity against the endogenous IRES. Spectrum of activity extends beyond IGF1R to include the c-myc IRES. The small molecule IRES inhibitor differentially modulates synthesis of the oncogenic (p64) and growth-inhibitory (p67) isoforms of Myc, suggesting that the IRES controls not only translational efficiency, but also choice of initiation codon. Sustained IRES inhibition has profound, detrimental effects on human tumor cells, inducing massive (>99%) cell death and complete loss of clonogenic survival in models of triple-negative breast cancer. The results begin to reveal new insights into the inherent complexity of gene-specific translational regulation, and the importance of IRES-mediated translation to tumor cell biology.

High expression of long intervening non-coding RNA OLMALINC in the human cortical white matter is associated with regulation of oligodendrocyte maturation.

BackgroundLong intervening non-coding RNAs (lincRNAs) are a recently discovered subclass of non-coding RNAs. LincRNAs are expressed across the mammalian genome and contribute to the pervasive transcription phenomenon. They display a tissue-specific and species-specific mode of expression and are present abundantly in the brain.ResultsHere, we report the expression patterns of oligodendrocyte maturation-associated long intervening non-coding RNA (OLMALINC), which is highly expressed in the white matter (WM) of the human frontal cortex compared to the grey matter (GM) and peripheral tissues. Moreover, we identified a novel isoform of OLMALINC that was also up-regulated in the WM. RNA-interference (RNAi) knockdown of OLMALINC in oligodendrocytes, which are the major cell type in the WM, caused significant changes in the expression of genes regulating cytostructure, cell activation and membrane signaling. Gene ontology enrichment analysis revealed that over 10% of the top 25 up- and down-regulated genes were involved in oligodendrocyte maturation. RNAi experiments in neuronal cells resulted in the perturbation of genes controlling cell proliferation. Furthermore, we identified a novel cis-natural antisense non-coding RNA, which we named OLMALINC-AS, which maps to the first exon of the dominant isoform of OLMALINC.ConclusionsOur study has demonstrated for the first time that a primate-specific lincRNA regulates the expression of genes critical to human oligodendrocyte maturation, which in turn might be regulated by an antisense counterpart.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-014-0091-9) contains supplementary material, which is available to authorized users.

Nutritional regulation of wound healing: the role of different amino acid mixture composition intake in wound repair. A preliminary study

The ability of the skin to repair wounds requires immediate changes in metabolism and expression and silencing of the genes controlling cell proliferation, migration, adhesion and dif- ferentiation. The energetic costs of wounds repair are enormous (1). Collagen represent around 50% of skin weight (about 20Kg), and synthesis costs are around 4 ATPs for any single amino acid (AA) inserted into the final composition of collagen (2). Moreover, the AA availability is indispensable to ensure the correct repair of the wound. Indeed, the poor state of dermal collagenous matrix is largely responsible for both the propensity to injury and slowness to heal observed in the elderly. To test whether different formulation of AA mixture could improve wound repair, we fed male rats (14 months old) with three balanced diet containing 1) 100% essential AA mixture (EAA); 2) EAA 10% plus 90% non-essential (NE) AA (deficient diet - D) and 3) EAA 40% plus NEAA 60% (casein like diet – CL) for 30 days. Then, four excisional wound (diameter 5mm) were surgically made on dorsal skin of each animal. After 3, 15 and 30 post wound days (pwd) the wound area were measured, collected and analyzed by Sirius Red and IHC methods for TGFβ1 and Collagen 1. The D diet reduced weight of the animals up to 30% in 60 days although there was an increase of >18% of spontaneous caloric introduction, whereas the EAA and CL diets increased the rat weight of 6% and 12% respectively. Three pwd, the injured area in EAA and CL diets was 92% and 98% respect to day 0 (100%); whereas it was larger (120%) in D diet. Fif- teen pwd, the wounds were completely closed with EAA diet, whereas with CL and D diets the open area were 14% and 45% respectively. Thirty pwd, all wounds were closed but large scars were observed in D diet. TGFβ1 was early and strongly expressed in D diet, whereas it was much less expressed in EAA and CL diets. Sirius-red staining showed that the collagen architecture was more organized with EAA diet and less organized with D diet. Collagen was strongly expressed 15 pwd in CL diet and less expressed in D and EAA diets, whereas 30 pwd an intense staining was observed only in EAA diet. Our data show for the first time that malnutrition, diet rich in NEAA, seriously affects metabolism, leading to rapid decay of the animals and slows down the wound closure, due to dermal collagenous matrix impairment and collagen production, and pos- sibly related to excessive TGFβ1 expression. On the contrary, EAA diet controls expression of TGFβ1 and collagen production, accelerating the wound repair and blunting scarring.

Cancer specificity of promoters of the genes controlling cell proliferation.

Violation of proliferation control is a common feature of cancer cells. We put forward the hypothesis that promoters of genes involved in the control of cell proliferation should possess intrinsic cancer specific activity. We cloned promoter regions of CDC6, POLD1, CKS1B, MCM2, and PLK1 genes into pGL3 reporter vector and studied their ability to drive heterologous gene expression in transfected cancer cells of different origin and in normal human fibroblasts. Each promoter was cloned in short (335-800 bp) and long (up to 2.3 kb) variants to cover probable location of core and whole promoter regulatory elements. Cloned promoters were significantly more active in cancer cells than in normal fibroblasts that may indicate their cancer specificity. Both versions of CDC6 promoters were shown to be most active while the activities of others were close to that of BIRC5 gene (survivin) gene promoter. Long and short variants of each cloned promoter demonstrated very similar cancer specificity with the exception of PLK1-long promoter that was substantially more specific than its short variant and other promoters under study. The data indicate that most of the important cis-regulatory transcription elements responsible for intrinsic cancer specificity are located in short variants of the promoters under study. CDC6 short promoter may serve as a promising candidate for transcription targeted cancer gene therapy.

Novel t(7;10)(p22;p24) along with NPM1 mutation in patient with relapsed acute myeloid leukemia

Chromosomal abnormalities/genetic mutations associated with hematological malignancies alter the structure and function of genes controlling cell proliferation and differentiation through multiple and complex pathways, resulting different clinical outcomes. This is a case study of a lady presented with acute myeloid leukemia (AML M1) at our center who relapsed 10 years after the induction therapy. Cytogenetic and molecular analyses were performed in this case at the time of relapse to find out the chromosomal abnormalities and genetic abnormalities like FMS-like tyrosine kinase (FLT3) and nucleophosmin (NPM1) mutation. The cytogenetic analysis of bone marrow established a novel translocation t(7;10) (p22;q24) in 100% of the cells analyzed. Phytohaemagglutinin (PHA)-stimulated blood culture also revealed the same abnormality. Apart from this, the molecular analysis showed NPM1 exon 12 (hotspot) mutation in this patient. This was the first report of novel chromosomal translocation in this subset of AML in which a new translocation along with NPM1 mutation was discussed.

MicroRNA-195 Inhibits the Proliferation of Human Glioma Cells by Directly Targeting Cyclin D1 and Cyclin E1

Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3′-untranslated regions (3′-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.

How a Blood Cell Intracellular Signal Keeps Blood Flowing

‘‘Epo,’’ or erythropoietin, is not just a performance-enhancing drug for endurance athletes. It is an essential hormone that regulates the rate of erythropoiesis, or creation of new red blood cells. Basal Epo signaling ensures that such cells are made constantly at a low level, to replace wornout cells. Certain stresses, such as hypoxia, increase Epo and lead to a steep increase in erythropoiesis, a phenomenon known to mountain climbers, who adapt to the low oxygen at high altitudes by packing more red blood cells into their blood. The Epo signaling cascade begins when the hormone binds to the Epo receptor. This ultimately leads to phosphorylation of a transcription factor called Stat5, which turns on a suite of genes controlling cell proliferation. A key question in understanding the Epo-Stat5 system is whether and how basal and stress-induced signaling differ. In a new study in this issue of PLOS Biology, Emelinda Porpiglia, Merav Socolovsky, and colleagues set out to answer that question. The authors used fluorescently labeled antibodies to tag phosphorylated Stat5 in erythropoeitic liver cells of mouse embryos. They also used a variety of cell surface markers to distinguish early versus late erythroblasts (cells that differentiate into red blood cells). They characterized three aspects of Stat5 signaling within Epostimulated cells: the median fluorescence from all cells within a subpopulation of erythroblasts, the fraction of those cells that were engaged in Stat5 signaling, and the level of phosphorylated Stat5 within signaling cells. They found that Stat5 signaling was highest in precursor cells, and declined with maturation. Furthermore, precursors exhibited an ‘‘analog,’’ graded increase in signaling in response to rising Epo levels. This graded increase was a combination of an increase in the number of signaling cells, and an increase in the signaling intensity within each cell. In contrast, older cells, which overall had much lower Stat5 signaling, responded to an increase in Epo by increasing the number of signaling cells, but within each cell, the signal intensity was binary, either ‘‘on’’ or ‘‘off,’’ with little change in intensity in the ‘‘on’’ position. What regulates this transition from analog to binary response? In large part, they found, the ability to mount a highintensity and graded response was due to larger amounts of Stat5 protein in earlier cells, rather than differences in Epo receptor level. When the authors added exogenous Stat5 to older cells, they too were able to signal at the higher level and in a graded way. Conversely, in cells with a mutant Epo receptor unable to bind Stat5, only basal-level, binary signaling was present, similar to the older cells. A binary signal, the authors argue, provides an ideal way to filter out the noise at low incoming Epo signal strength, while the analog response at higher Epo concentrations provides the cell with a more dynamic ability to respond to varying levels of stress. Additionally, they found, part of the stress response was an increased production of transferrin receptor (CD71), probably to help bring in extra iron to make more hemoglobin. Understanding this system is important not only for a better theoretical understanding of the dynamics of cell signaling. Targeting the high-level Stat5 signal, while maintaining the low-level one, may be a viable strategy for treating myeloproliferative disorders, including some types of leukemia, in which Stat5 is constitutively active.

Abstract 2193: WT1 mediated regulation of growth control genes in leukemia

Abstract Prognosis and therapy of acute leukemia is influenced by leukemia-specific genetic alterations, thus highlighting the importance of identifying novel prognostic markers. One such potential marker, the Wilms’ tumor gene (WT1), a zinc finger transcription factor, is expressed in leukemic blasts and has been found to be mutated in approximately 10 percent of leukemia cases. Work in other tumor systems has shown that WT1 up-regulates expression of genes promoting cancer progression, including the angiogenic and mitogenic factor VEGF. Microarray studies have also correlated expression of JAG1 (Jagged 1) and CCNA1 (Cyclin A) with WT1 in Acute Myeloid Leukemia (AML) samples. We have identified potential WT1 binding sites within the promoters of both JAG1 and CCNA1 and have compared the expression of these genes to that of WT1, using real time quantitative PCR (QRTPCR), in pediatric leukemia and normal bone marrow. Compared to normal bone marrow, we observed lower than normal levels of WT1 in a majority of pediatric ALL samples, associated with lower than normal levels of VEGF, JAG-1 and CCNA-1. Conversely in pediatric AML(M3) we observed elevated levels of WT1 associated with elevated JAG-1 and CCNA-1. Mutations of the WT1 zinc finger (ZF) DNA binding domain have also been described in poor prognosis leukemias. To identify novel ZF mutations we sequenced WT1 in twelve pediatric acute leukemia samples. No ZF domain mutations were identified among these samples with high WT1 expression. However, a well-described SNP (rs 16754, also in exon 7), identified as a good prognostic marker in Cytogenetically Normal AML, was observed either as a homozygous or heterozygous variant of the WT1 gene. Because a majority of WT1 mutations identified in leukemias are frameshift mutations leading to a truncated protein lacking the ZF DNA binding domain, we created a truncation mutant of isoform A-WT1 lacking the ZF domain and assessed its function in K562 cells. Using QRTPCR we quantified the effect of over-expression of wild type and mutant WT1 on VEGF, JAG1 and CCNA1 expression. Compared to the vector control, wild type WT1 upregulated levels of JAG1 and CCNA1 but not VEGF mRNA. Mutant WT1 did not upregulate JAG1 or CCNA1. Our data and other studies have shown that JAG1 is elevated in AML patient samples, implicating the NOTCH 1 pathway that promotes cell proliferation and blocks differentiation. Thus, these results suggest that WT1 may act as an oncogene in K562 cells, in part by upregulating the growth promoting NOTCH-1 ligand, JAG1. Similarly, cell cycle regulation by WT1, demonstrated in other systems, may be mediated by transcriptional regulation of CCNA1. WT1 may contribute to leukemogenesis through transcriptional regulation of genes controlling cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2193. doi:1538-7445.AM2012-2193