Unscheduled Expression Research Articles

Nizp1 is a specific NUP98-NSD1 functional interactor that regulates NUP98-NSD1-dependent oncogenic programs.

NSD1, NSD2 and NSD3 proteins constitute a family of histone 3 lysine 36 (H3K36) methyltransferases with similar domain architecture, but diversified activities, in part, dependent on their non-enzymatic domains. These domains, despite their high sequence identity, recruit the hosting proteins to different chromatin regions through the recognition of diverse epigenetic marks and/or associations to distinct interactors. In this sense, the PHDvC5HCH finger tandem domain represents a paradigmatic example of functional divergence within the NSD family. In this work, we prove and give a structural rationale for the uniqueness of the PHDvC5HCH domain of NSD1 in recognizing the C2HR Zinc finger domain of Nizp1 (NSD1 interacting Zn finger protein). Importantly, we show that, in a leukaemogenic context, Nizp1 is pivotal in driving the unscheduled expression of HoxA genes and of genes involved in the type I IFN pathway, triggered by the expression of the fusion protein NUP98-NSD1. These data provide the first insight into the pathophysiological relevance of the Nizp1-NSD1 functional association. Targeting of this interaction might open new therapeutic windows to inhibit the NUP98-NSD1 oncogenic properties.

Bloom syndrome DNA helicase deficiency is associated with oxidative stress and mitochondrial network changes

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.

Context-Dependent Tumorigenic Effect of Testis-Specific Mitochondrial Protein Tiny Tim 2 in Drosophila Somatic Epithelia.

We have undertaken a study towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. Here, we show that in the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. Our results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context.

Krüppel-like factor 4 (KLF4) regulates the miR-183~96~182 cluster under physiologic and pathologic conditions

MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that post-transcriptionally control the translation and stability of target mRNAs in a sequence-dependent manner. MiRNAs are essential for key cellular processes including proliferation, differentiation, cell death and metabolism, among others. Consequently, alterations of miRNA expression contribute to developmental defects and a myriad of diseases.The expression of miRNAs can be altered by several mechanisms including gene copy number alterations, aberrant DNA methylation, defects of the miRNA processing machinery or unscheduled expression of transcription factors. In this work, we sought to analyze the regulation of the miR-182 cluster, located at the 7q32 locus, which encodes three different miRNAs that are abundantly expressed in human embryonic stem cells and de-regulated in cancer. We have found that the Krüppel-like factor 4 (KLF4) directly regulates miR-182 cluster expression in human embryonic stem cells (hESCs) and in melanoma tumors, in which the miR-182 cluster is highly expressed and has a pro-metastatic role. Furthermore, higher KLF4 expression was found to be associated with metastatic progression and poor patient outcome. Loss of function experiments revealed that KLF4 is required for melanoma cell maintenance. These findings provide new insights into the regulation of the miR-182 cluster expression and new opportunities for therapeutic intervention in tumors in which the KLF4-miR-182 cluster axis is deregulated.

Endometriosis as systemic genetic problem

Current data contributing to the origin and development of endometriosis (E) — sever common disease of reproductive age women are reviewed. Numerous theories and hypothesis as well as basic pathological mechanisms underlying E are briefly outlined. Whole gene-net and particular groups of candidate genes involved in E are presented. The later include genes of detoxification system, sex hormones and their receptors, cytokines gene, onco- suppressors the genes contributing to embryonic development of female reproductive organs and cell proliferation Major molecular genetic data imply epigenetic impact in the origin of disease which results from progressive disregulation of endometrium genome functions provoked by unfavorable combinations of candidate genes of some signaling pathways and defects in their expression due to abnormal methylation pattern. Histone modifications and the faults of miRNA content. In line with systemic genetic concept initial stages of E could be provoked by different genetic, epigenetic and even external factors which outcome is the same pathophysiological and pathomorphological damages clinically classified as E. Two feasible molecular genetic routs of E. are suggested. Both are relying on unscheduled expression of early embryonic genes such as WNT4, HOX10, HOX11, GALT provoking the changes in genetic program of endometriotic cells (metaplasia) and their subsequent progression as the lesions of ectopic endometrium

Mitochondrial hyperfusion induced by loss of the fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress

Mitochondrial fission and fusion cycles are integrated with cell cycle progression. In this paper, we demonstrate that the inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by a hyperfused mitochondrial structure and unscheduled expression of cyclin E in the G2 phase. This persistent replication stress then induces an ATM-dependent activation of the G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and reactive oxygen species (ROS) generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates the DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics.

Eph receptor function is modulated by heterooligomerization of A and B type Eph receptors

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph-Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.

Abstract 4287: Ets-related gene (Erg) expression in developing and adult mouse tissues

Abstract Oncogenic activation of the ETS Related Gene (ERG) in human neoplasms was originally identified in subsets of Ewing sarcomas, myeloid leukemias and, recently, in the majority of prostate cancers. Expression of human ERG protein and consequently its functions in normal and disease states needs to be better understood in light of its suggested role in cell differentiation and proliferation. In this study, we analyzed temporal and spatial expression of the Erg protein by immunohistochemical analysis during mouse embryonic and adult organogenesis using a highly specific ERG monoclonal antibody (CPDR ERG-MAb) developed by our group. Initially, we evaluated the specificity of three recently available ERG monoclonal antibodies (Epitomics EPR 3864 and EPR 3863, and CPDR ERG-MAb) using ERG expressing MOLT4, KG1, COLO 320, VCaP tumor cell lines and LNCaP, MCF7, Jurkat cell lines that do not express ERG. Unlike, rabbit monoclonal antibodies to ERG obtained from Epitomics, the ERG MAb did not show cross reactivity to FLI-1 or to the other ETS related proteins. Our comprehensive evaluation of mouse tissues established a widespread immunolocalization of Erg protein in endothelial cells and restricted expression in precartilage and hematopoietic tissues. Intriguingly, Erg is not expressed in any epithelial tissue including prostate epithelium, a common site of tumors with ERG rearrangements and unscheduled ERG expression. Further, use of two different monoclonal antibodies revealed that infiltrating lymphocytes that are occasionally seen in the prostate environment were positive for Fli-1, did not exhibit any Erg expression. These findings will further aid in investigations of Erg functions in normal and disease conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4287. doi:10.1158/1538-7445.AM2011-4287

Setting and resetting of epigenetic marks in malignant transformation and development

Epigenetic modifications, such as DNA methylation and post-translation modifications of histones, have been shown to play an important role in chromatin structure, promoter activity, and cellular reprogramming. Large protein complexes, such as Polycomb and trithorax, often harbor multiple activities which affect histone tail modification. Nevertheless, the mechanisms underlying the deposition of these marks, their propagation during cell replication, and the alteration on their distribution during transformation still require further study. Here we review recent data on those processes in both normal and cancer cells, and we propose that the unscheduled expression of oncogenic transcription factors causes reprogramming of normal cells into cancer stem cells.

1304 DOWN-REGULATION OF ERG IN PROSTATE CANCER CELLS ACTIVATES EPITHELIAL CELL DIFFERENTIATION

You have accessJournal of UrologyProstate Cancer: Basic Research VII1 Apr 20101304 DOWN-REGULATION OF ERG IN PROSTATE CANCER CELLS ACTIVATES EPITHELIAL CELL DIFFERENTIATION Shyh-Han Tan, Nichelle Shah, Ahmed Mohamed, Taduru Sreenath, Gyorgy Petrovics, Albert Dobi, David G. McLeod, and Shiv Srivastava Shyh-Han TanShyh-Han Tan More articles by this author , Nichelle ShahNichelle Shah More articles by this author , Ahmed MohamedAhmed Mohamed More articles by this author , Taduru SreenathTaduru Sreenath More articles by this author , Gyorgy PetrovicsGyorgy Petrovics More articles by this author , Albert DobiAlbert Dobi More articles by this author , David G. McLeodDavid G. McLeod More articles by this author , and Shiv SrivastavaShiv Srivastava More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.888AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The prostate epithelium consists of luminal and basal cell types. The differentiation from the phenotype of basal cell towards luminal cell can be detected by the expression of specific protein markers: basal epithelial cells express CK-5, CK-14 and p63; luminal epithelial cells express secretory proteins such as PSA, SLC45A3 (prostein) and MSMB (PSP94). Frequent occurrence of the TMPRSS2-ERG gene fusions (>60%) in prostate cancer leads to androgenic induction of the ERG mRNA and protein in tumor cells. We hypothesize that the deregulation of ERG protein expression negatively affects some aspects prostate epithelial differentiation. We aim to understand the function of ERG in the regulation of signaling pathways that affect differentiation of prostate epithelium. METHODS Prostate cancer cells, VCaP which over-express ERG were used in 2-D and 3-D cell culture model systems. We used siRNA and shRNA to deplete the ERG proteins in these cells. The expression of various structural and secretory differentiation markers were examined to by QRT-PCR, Western blots and immuno-fluorescence assays to detect the changes in cell differentiation. The putative promoters that are regulated by ERG were examined by chromatin immuno-precipitation experiments. RESULTS We show that the depletion of ERG by siRNA methods induce changes in cell morphology, concomitant down-regulation of C-MYC and over-expression of cell differentiation markers such as PSA, SLC45A3 and MSMB. Marker of inter-cellular tight junctions, ZO-1 was significantly altered in VCaP cells in response to ERG knock down. Morphology similar to more organized epithelial cellular phenotype was evident in 3-D cultures of VCaP with ERG knockdown. CONCLUSIONS This study suggests that unscheduled expression of ERG oncoprotein in prostate epithelial cells as a result of the fusion of TMPRSS2 promoter to the ERG protein coding sequence may abrogate cell differentiation program favoring cell proliferation. A better understanding of the ERG function in prostate epithelial cell differentiation could lead to the development novel therapeutic strategies for prostate cancer. Rockville, MD© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e504 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shyh-Han Tan More articles by this author Nichelle Shah More articles by this author Ahmed Mohamed More articles by this author Taduru Sreenath More articles by this author Gyorgy Petrovics More articles by this author Albert Dobi More articles by this author David G. McLeod More articles by this author Shiv Srivastava More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Unscheduled expression of CDC25B in S-phase leads to replicative stress and DNA damage

BackgroundCDC25B phosphatase is a cell cycle regulator that plays a critical role in checkpoint control. Up-regulation of CDC25B expression has been documented in a variety of human cancers, however, the relationships with the alteration of the molecular mechanisms that lead to oncogenesis still remain unclear. To address this issue we have investigated, in model cell lines, the consequences of unscheduled and elevated CDC25B levels.ResultsWe report that increased CDC25B expression leads to DNA damage in the absence of genotoxic treatment. H2AX phosphorylation is detected in S-phase cells and requires active replication. We also report that CDC25B expression impairs DNA replication and results in an increased recruitment of the CDC45 replication factor onto chromatin. Finally, we observed chromosomal aberrations that are also enhanced upon CDC25B expression.ConclusionOverall, our results demonstrate that a moderate and unscheduled increase in CDC25B level, as observed in a number of human tumours, is sufficient to overcome the S-phase checkpoint efficiency thus leading to replicative stress and genomic instability.

Ultraviolet light induces the sustained unscheduled expression of cyclin E in the absence of functional p53

Cell cycle progression is regulated through changes in the activity of cyclin-dependent kinases that are, in turn, regulated by the expression of their respective cyclin partners. In primary cells, cyclin E expression increases through the G1 phase of the cell cycle and peaks near the G1/S boundary. The unscheduled expression of cyclin E in primary human fibroblasts leads to chromosomal instability that is greatly increased by loss of the p53 tumour suppressor. Intriguingly, ultraviolet light (UV), the most prevalent environmental carcinogen, is similarly known to induce chromosomal instability more dramatically in the absence of p53. Here we report that UV light transiently increased the expression of cyclin E in normal human fibroblasts. Strikingly, cyclin E levels remained elevated for an extended period of time in the absence of functional p53. UV-induced cyclin E expression was not restricted to the G1/S boundary but remained elevated throughout S phase and this correlated with a massive accumulation of p53-deficient fibroblasts in this phase of the cell cycle. Forced expression of cyclin E alone was insufficient to cause a similar S phase arrest but forced expression of cyclin E led to an increase in the proportion of UV-irradiated cells in S phase. The present work suggests that p53 affects S phase progression following UV exposure by preventing the sustained unscheduled expression of cyclin E and that this may limit the clastogenic and carcinogenic effects of UV light.

Mouse Cofactor of BRCA1 (Cobra1) Is Required for Early Embryogenesis

BackgroundNegative elongation factor (NELF) is a four-subunit protein complex conserved from Drosophila to humans. In vitro biochemical and tissue culture-based studies have demonstrated an important role of NELF in controlling RNA polymerase II (Pol II) pausing in transcription. However, the physiological significance of NELF function is not clear due to the lack of any genetic systems for studying NELF.Principal FindingsHere we show that disruption of the mouse B subunit of NELF (NELF-B), also known as cofactor of BRCA1 (Cobra1), causes inner cell mass (ICM) deficiency and embryonic lethality at the time of implantation. Consistent with the phenotype of the Cobra1 knockout (KO) embryos, knockdown of Cobra1 in mouse embryonic stem cells (ESCs) reduces the efficiency of colony formation and increases spontaneous differentiation. Cobra1-depleted ESCs maintain normal levels of Oct4, Nanog, and Sox2, master regulators of pluripotency in ESCs. However, knockdown of Cobra1 leads to precocious expression of developmental regulators including lymphoid enhancer-binding factor 1 (Lef1). Chromatin immunoprecipitation (ChIP) indicates that Cobra1 binds to the Lef1 promoter and modulates the abundance of promoter-bound RNA polymerase.Conclusions Cobra1 is essential for early embryogenesis. Our findings also indicate that Cobra1 helps maintain the undifferentiated state of mESCs by preventing unscheduled expression of developmental genes.

New Insights on the Perturbations of T Cell Cycle During HIV Infection

The role of the Human Immunodeficiency Virus (HIV) in the pathogenesis of the Acquired Immune-Deficiency Syndrome (AIDS) is changed. Direct HIV-mediated killing of CD4(+) T cells is not the only mechanism leading to lymphocyte depletion. There is increasing evidence that, during the chronic phases of infection, T cell activation, accelerated cell turnover, and cytokines imbalance induce the so-called cell cycle dysregulation (CCD). CCD is a recently discovered immune-pathogenic mechanism that mainly induces the depletion of both CD4(+) and CD8(+) uninfected T cells. It is due to a significant perturbation of protein metabolism as ubiquitin pathway defects of protein degradation are associated with an increased and unscheduled expression of cyclin B and p34 cdc kinase. Moreover, significant changes in the nucleolar structure and post-translational regulation of nucleolin have also been described. As modulation of CCD by anti-retroviral and immune-therapies has been suggested to predict a good immunological response in HIV-infected patients, a better understanding of such a mechanism is needed in order to further clarify its role in the pathogenesis and progression of HIV infection.

Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage

Several methods to synchronize cultured cells in the cell cycle are based on temporary inhibition of DNA replication. Previously it has been reported that cells synchronized this way exhibited significant growth imbalance and unscheduled expression of cyclins A and B1. We have now observed that HL-60 cells exposed to inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea), at concentrations commonly used to synchronize cell populations, had histone H2AX phosphorylated on Ser-139. This modification of H2AX, a marker of DNA damage (induction of DNA double-strand breaks; DSBs), was most pronounced in S-phase cells, and led to their apoptosis. Thus, to a large extent, synchronization was caused by selective kill of DNA replicating cells through induction of replication stress. In fact, similar synchronization has been achieved by exposure of cells to the DNA topoisomerase I inhibitor camptothecin, a cytotoxic drug known to target S-phase cells. A large proportion of the surviving cells 'synchronized' by DNA replication inhibitors at the G1/S boundary had phosphorylated histone H2AX. Inhibitors of DNA replication, thus, not only selectively kill DNA replicating cells, induce growth imbalance and alter the machinery regulating progression through the cycle, but they also cause DNA damage involving formation of DSBs in the surviving ('synchronized') cells. The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication.

A Novel Three-parameter Flow Cytometric Analysis for Cell Cycle

Objective: To set up a three-parameter method for cell cycle analysis by two-laser flow cytometer, which can detect two types of cyclin plus DNA content in one measurement, and that analyze unscheduled expression of cyclins. Methods: Three-color fluorescence was used for analysis of two types of cyclins and DNA content simultaneously in individual cells by two-laser flow cytometry. MOLT-4 cells were used to study the expression of major cyclins in mammalian cells. A Triton-X100 permeabilization procedure was optimized for detection of two types of cyclins. One cyclin was stained directly with a FITC-conjugated monoclonal antibody (mAb), and the other, indirectly with RPE-Cy5-conjugated secondary antibody, while DNA was stained with the fluorochrome DAPI. mAMSA and mimosine treated MOLT-4 cells were used to test this three-parameter method. Results: Permeabilization with 0.5% Triton-X100 in PBS containing 1% BSA for 5 min on ice provided optimal conditions for the simultaneous labelling of two cyclins plus DNA in single cells. It was found that the emission spectrum of the three dyes (DAPI, FITC and RPE-Cy5) could be measured with no compensation. Based on cyclinA/cyclinE/DNA flow cytometric analysis, asynchronously growing MOLT-4 cells could be divided into 6 compartments (G1o, G1e, G1l, S, G2, and M) simultaneously, allowing for analysis of cell cycle phase specific perturbations without the necessity of cell synchronization. Unscheduled cyclin B1 expression was observed in G1 cells treated with mimosine and cyclin E in G2 cells treated with mAMSA. We found that unscheduled cyclin expression paralleled expected cyclin expression. Conclusion: Thus, three-color FCM analysis of cells may not only be applied to measure unscheduled vs. expected cyclin expression but may also be used to estimate the fraction of cycling cells in up to 6 cell populations.

Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster.

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.

Preliminary characterisation ofDML1, an essentialSaccharomyces cerevisiaegene related tomisatoofDrosophila melanogaster

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1v heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho 0 ] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement. 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

High frequency of Ikaros isoform 6 expression in acute myelomonocytic and monocytic leukemias: implications for up-regulation of the antiapoptotic protein Bcl-XL in leukemogenesis

While studying Ikaros proteins in childhood acute myeloid leukemia (AML), Ikaros isoform 6 (Ik6) expression was detected in 7 of 10 cases of M4 and M5 leukemia, but in none of the remaining French-American-British subtypes (M2, 8 cases; M7, 6 cases). The spliced Ikaros isoforms 4 to 8 (Ik4-8) suppress the function of full-length Ik1 or Ik2 in a dominant-negative manner, owing to their reduced numbers of DNA binding sites. Thus, dominant-negative Ikaros isoforms may inhibit the normal transcriptional regulation of hematopoietic cell development. To clarify the function of Ik6 in developing blood cells, this isoform was transiently transfected into an Ik2(+), interleukin-3 (IL-3)-dependent 32D murine myeloid precursor cell line and studied the expression of Bcl-2 family proteins in relation to in vitro cell growth, using a tetracycline-inducible TREx system. The possibility of aberrant cell regulation due to Ikaros functional changes was examined by cotransfecting both Ik2 and Ik6 into Ikaros/Aiolos/Helios triple-negative Cos-7 cells. The results demonstrated IL-3-independent growth by Ik6-transfected 32D clones coincident with up-regulation of the antiapoptotic protein Bcl-XL. Up-regulation of Bcl-XL, but not of other Bcl-2 family proteins, was associated with the suppression of functional Ik2 by Ik6 in a dominant-negative fashion. Thus, the pathogenesis of myelomonocytic/monocytic AML may involve aberrant regulation of apoptosis due to unscheduled expression of the Ik6 isoform.

Unscheduled expression of capsid protein IIIa results in defects in adenovirus major late mRNA and protein expression

Adenovirus gene expression is to a large extent regulated at the level of alternative RNA splicing. For example, in the major late region 1 (L1) unit, a common 5′ splice site can be joined to two alternative 3′ splice sites, resulting in the formation of the so-called 52,55K (proximal 3′ splice site) or the IIIa (distal 3′ splice site) mRNAs. Whereas, the 52,55K mRNA is expressed both early and late during infection, the IIIa mRNA is strictly confined to the late phase of the infectious cycle. We have previously shown that IIIa mRNA splicing is subjected to a tight viral control of IIIa 3 splice site usage. In an attempt to determine why adenovirus uses elaborate mechanisms to confine IIIa mRNA production to the late phase of infection, we characterized the phenotype of a recombinant adenovirus expressing the IIIa protein from an inducible tetracycline regulated gene cassette. The results show that expression of the IIIa protein during the early phase of infection results in a significant reduction in late viral protein synthesis and a moderate block to viral DNA replication. Interestingly, unscheduled IIIa protein expression resulted in a perturbation of the accumulation of alternatively spliced L1 mRNAs. Thus, 52,55K mRNA accumulation was inhibited while no effects on endogenous IIIa mRNA expression was detected.