Genetic driver mutations define the expression signature and microenvironmental composition of high-grade gliomas.

High-grade gliomas (HGG), including glioblastomas, are characterized by invasive growth, resistance to therapy, and high inter- and intra-tumoral heterogeneity. The key histological hallmarks of glioblastoma are pseudopalisading necrosis and microvascular proliferation, which allow pathologists to distinguish glioblastoma from lower-grade gliomas. In addition to being genetically and molecularly heterogeneous, HGG are also heterogeneous with respect to the composition of their microenvironment. The question of whether this microenvironmental heterogeneity is driven by the molecular identity of the tumor remains controversial. However, this question is of utmost importance since microenvironmental, non-neoplastic cells are key components of the most radiotherapy- and chemotherapy-resistant niches of the tumor. Our work demonstrates a versatile, reliable, and reproducible adult HGG mouse model with NF1-silencing as a driver mutation. This model shows significant differences in tumor microenvironment, expression of subtype-specific markers, and response to standard therapy when compared to our established PDGFB-overexpressing HGG mouse model. PDGFB-overexpressing and NF1-silenced murine tumors closely cluster with human proneural and mesenchymal subtypes, as well as PDGFRA-amplified and NF1-deleted/mutant human tumors, respectively, at both the RNA and protein expression levels. These models can be generated in fully immunocompetent mixed or C57BL/6 genetic background mice, and therefore can easily be incorporated into preclinical studies for cancer cell-specific or immune cell-targeting drug discovery studies.

Chromatin remodeling and circadian control: master regulator CLOCK is an enzyme.

The molecular machinery that governs circadian rhythmicity is based on clock gene products organized in regulatory feedback loops. Recently, we have shown that CLOCK, a master circadian regulator, has histone acetyltransferase activity essential for clock gene expression. The Lys-14 residue of histone H3 is a preferential target of CLOCK-mediated acetylation. As the role of chromatin remodeling in eukaryotic transcription is well recognized, this finding identified unforeseen links between histone acetylation and cellular physiology. Indeed, we have shown that the enzymatic function of CLOCK drives circadian control. We reasoned that CLOCK's acetyltransferase activity could also target nonhistone proteins, a feature displayed by other HATs. Indeed, CLOCK also acetylates a nonhistone substrate: its own partner, BMAL1. This protein undergoes rhythmic acetylation in the mouse liver, with a timing that parallels the down-regulation of circadian transcription of clock-controlled genes. BMAL1 is specifically acetylated on a unique, highly conserved Lys-537 residue. This acetylation facilitates recruitment of the repressor CRY1 to BMAL1, indicating that CLOCK may intervene in negative circadian regulation. Our findings reveal that the enzymatic interplay between two clock core components is crucial for the circadian machinery.

Lymphocytic choriomeningitis virus mx strain does not induce the expression of tumor-associated carbonic anhydrase IX in persistently infected HeLa cells.

Summary. - Lymphocytic choriomeningitis virus (LCMV) is an arenavirus that readily causes persistent infections, in which it does not interfere with vital functions of the cells, but can affect expression of "luxurious" genes. MX strain of LCMV (MX LCMV) has been identified as an agent transmissible by cell-to-cell contact in the human carcinoma MaTu cells grown in a mixed culture with HeLa cells. When compared to uninfected HeLa, the MaTu-MX-infected HeLa cells, to which the virus was transmitted via co-cultivation with mitomycin C-treated MaTu cells, showed an elevated expression of a protein called MN, suggesting that MN can be induced by MX LCMV. MN protein was later identified as the carbonic anhydrase isoform IX (CA IX), whose expression has been predominantly associated with hypoxic tumors of poor prognosis. Since the proposal that MX LCMV can induce such a cancer-related protein could substantially change our view on the biology of LCMV-host interaction we undertook its verification. Instead of co-cultivation, we used MaTu cell-free extracts to transmit MX LCMV to HeLa cells. These cells were then grown for more than 30 passages, but the level of MN/CA IX did not increase throughout the whole cultivation period as compared to uninfected HeLa cells. Moreover, a treatment of MaTu-MX-infected HeLa cells with ribavirin eliminated the virus, but did not reduce the MN/CA IX expression. Our results clearly showed that MN/CA IX is independent of MX LCMV and that the virus itself does not influence the MN/CA IX level in HeLa cells.

Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors.

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.

Block-replacement mutagenesis for functional dissection of multiple transcription factor complexes.

We propose a simple method for investigation of roles of individual transcription factors in complexes of multiple factors bound to the same cis element. By block-replacement mutagenesis, the whole cis element is replaced with a new one containing a binding site for a single factor. From the reporter activity of the mutant promoter construct, the importance of the individual factor in transcriptional activation is deduced. This approach allowed us to functionally identify SP1 as the most important PR1 binding transcription factor in the MN promoter.

P53 tumour suppressor modulates transcription of the TATA-less gene coding for the tumour-associated carbonic anhydrase MN/CA IX in MaTu cells.

MN/CA IX (MN) exhibits a strong association with tumours. Co-transfection experiments revealed that in MaTu cells the activity of the (-173;+31) MN promoter construct was repressed by the wild type p53 in a dose-responsive manner and stimulated by the (143(Val-->Ala)) mutant. Upregulation of endogenous p53 by mitomycin C treatment in MaTu cells also had a profound effect on MN expression as well as the activity of MN promoter in a reporter construct. p53 can thus modulate MN expression and at least in a subset of tumours the changed p53 status might be responsible for MN positivity. Co-transfections with internally deleted MN promoter constructs demonstrated that the wild type p53 exerts its repression activity on the level of the basal transcriptional machinery and not on a particular cis element within the MN promoter.

Acute effects of interferon on estrogen receptor function do not involve the extracellular signal-regulated kinases p42mapk and p44mapk.

Exposure to type I interferons (IFN) increased estrogen receptor (ER) ligand binding and induced protein kinase C (PKC) translocation within 30 min but had no effect on net incorporation of [32P] into ER in Madin Darby bovine kidney (MDBK) cells. Ligand binding was also increased within 30 min by phorbol ester and the protein phosphatase inhibitor okadaic acid. Mitogen-activated protein (MAP) kinase phosphorylation was initially inhibited between 2 and 30 min and subsequently activated between 30 and 60 min after treatment with IFN. The activatory response was blocked by the PKC inhibitor Ro 31-8220. Following transient transfection with an ERE-CAT reporter construct, IFN increased CAT expression after 6 h but decreased ER ligand binding, transcriptional activity and phosphorylation after 48 h, probably as a result of decreased ER concentrations. The results rule out rapid activation of ER ligand binding through phosphorylation at Ser118 by MAP kinase because (1) the increase in ligand binding preceded activation of MAP kinase, and (2) IFN had no short-term effect on [32P]incorporation or ER transcriptional activity. The rapid effect of IFN on ER ligand binding is postulated to reflect phosphorylation of the receptor at Tyr537 by p56lck, a member of the Src family of PKC-activated tyrosine kinases.

Transcriptional regulation of the MN/CA 9 gene coding for the tumor-associated carbonic anhydrase IX. Identification and characterization of a proximal silencer element.

The MN/CA 9 (MN) gene encodes a tumor-associated isoenzyme of the carbonic anhydrase family. Functional characterization of the 3. 5-kilobase pair MN 5' upstream region by deletion analysis led to the identification of the -173 to +31 fragment as the MN promoter. In vitro DNase I footprinting revealed the presence of five protected regions (PRs) within the MN promoter. Detailed deletion analysis of the promoter identified PR1 and PR2 (numbered from the transcription start) as the most critical for transcriptional activity. PR4 negatively affected transcription, since its deletion led to increased promoter activity and was confirmed to function as a promoter-, position-, and orientation-independent silencer element. Mutational analysis indicated that the direct repeat AGGGCacAGGGC is required for efficient repressor binding. Two components of the repressor complex (35 and 42 kDa) were found to be in direct contact with PR4 by UV cross-linking. Increased cell density, known to induce MN expression, did not affect levels of PR4 binding in HeLa cells. Significantly reduced repressor level seems to be responsible for MN up-regulation in the case of tumorigenic CGL3 as compared with nontumorigenic CGL1 HeLa x normal fibroblast hybrid cells.

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.

Identification of MaTu-MX agent as a new strain of lymphocytic choriomeningitis virus (LCMV) and serological indication of horizontal spread of LCMV in human population.

In this study we elucidated the molecular character of MaTu-MX, previously described as an unusual transmissible agent. Amino acid sequencing of peptides generated from a 58-kDa MX-related protein purified from MaTu human carcinoma cells allowed us to identify it as a nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV). Northern blot analysis detected LCMV-specific RNAs in MaTu cells. Comparative immunoprecipitations showed cross-reactivity between NP of LCMV strain WE and MX NP. Using RT-PCR, we have cloned MX NP cDNA. According to sequence comparison, MX LCMV is as closely related to both LCMV strains WE and Armstrong as these strains are to one another. Based on this finding we propose that MX is a new strain of LCMV. We also showed that the stability of MX NP in MaTu cells is very high and that the virus is transmissible by cell-to-cell contact or by cell-free extract to human HeLa and monkey Vero cells, but not to human AGS, canine MDCK, mouse NIH 3T3, and hamster CHO cells. Finally, employing MX LCMV NP in immunoprecipitation and solid-phase radioimmunoassay, we found 37.5% prevalence of anti-LCMV antibodies in human sera, suggesting possible horizontal spread of the virus in the human population.

Up-regulation of p53 by antisense expression of HPV18 E6 oncogene does not influence the level of MN/CA IX tumor-associated protein in HeLa cervical carcinoma cells.

Oncogenic potential of human papillomaviruses is related to capacity of HPV-encoded oncoproteins to bind and inactivate tumor suppressor proteins. Interaction of p53 with HPV E6 results in aberrant regulation of various cellular genes. We evaluated the possible involvement of MN/CA9 gene, whose expression is closely associated with cervical carcinomas, in regulatory pathways driven by p53 and E6. We demonstrated that one of the two p53 consensus sequences present in MN/CA9 promoter participates in DNA-protein interaction but it does not bind p53. Tetracycline-inducible antisense expression of HPV18 E6 in human cervical carcinoma HeLa cells resulted in increased level of p53 but did not affect expression of MN/CA IX protein. Therefore we conclude that at least in HeLa cells there is no direct relationship between expression of MN/CA IX and expression of E6 or p53.

Sequencing analysis of prion genes from red deer and camel.

An abnormal isoform of the prion protein (PrP) appears to be the agent responsible for transmissible spongiform encephalopathies (TSE). The normal isoform of PrP is host-encoded and expressed in the central nervous system. The recent bovine spongiform encephalopathy (BSE) epidemic in the UK and the incidence of prion-related diseases in other animals could indicate that ruminants are highly susceptible to infection via ingestion of prion-contaminated food. Sequence analysis of PrP gene open reading frames from red deer and camel was carried out to investigate sequence variability of these genes among ruminants.

Biology of tick-borne encephalitis virus.

Tick-borne encephalitis (TBE) virus is an important human pathogen belonging to the genus Flavivirus within the family Flaviviridae. The genome of the TBE virus is a single-stranded RNA (ssRNA) molecule of positive polarity encoding all the viral proteins within a single open reading frame (ORF). TBE virus shares common physical and genetic characteristic of the flavivirus genus. Two subtypes of the TBE virus have been described: (1) European, endemic in many parts of Europe and transmitted by Ixodes ricinus ticks, and (2) Far Eastern (Russian spring summer encephalitis (RSSE) virus), endemic in Far East and transmitted by Ixodes persulcatus ticks.

Structure of an ovine interferon receptor and its expression in endometrium.

A sheep type I interferon receptor (oIFNAR1) cDNA was isolated from a lambda-ZAP library using a reverse transcription (RT)-PCR product probe generated from oestrous endometrial RNA. The oIFNAR1 cDNA was 79, 66 and 95% homologous to human, murine and bovine IFNAR1 cDNAs respectively. The encoded receptor was a 560-amino acid transmembrane protein 80, 66 and 95% similar to human, murine and bovine IFNAR1 respectively. Northern blot analysis of endometrial mRNA revealed the presence of 6.5, 4.3 and 3.7 kb transcripts. Using semi-quantitative RT-PCR the oIFNAR1 mRNA was not found to be down-regulated after 72 h treatment with bovine recombinant IFN-alpha I in in vitro experiments with endometrial explants.

Application of suppression PCR to the megaprimer method for site-directed mutagenesis.

Combination of suppression polymerase chain reaction (PCR) and megaprimer method of site-directed mutagenesis allows specific reamplification of extended megaprimer. This modification is proposed for templates that are difficult to amplify when standard megaprimer procedures do not generate sufficient yield of mutated product.

Heterogeneity in the third intracytoplasmic region of the oxytocin receptor-encoding gene.

The oxytocin receptor (OTR), a member of the seven-transmembrane domain guanine-nucleotide-binding protein (G protein) coupled receptor family plays a central role in lactation, ovarian cyclicity and reproductive behaviour. Recent cloning and sequencing unexpectedly revealed that the third intracytoplasmic region (3ICR) of the sheep receptor has 3 and 2 additional amino acids (aa) relative to the rat and human receptors, respectively. We have now confirmed, by sequencing polymerase chain reaction (PCR)-derived genomic fragments coding for the OTR 3ICR from a variety of ruminant and non-ruminant species, that additional aa are a general phenomenon in ruminants.

Nucleotide sequence of the protein E gene of the tick-borne encephalitis virus strain 595 isolated in Slovakia.

Tick-borne encephalitis (TBE) virus, strain 595 was isolated from Ixodes ricinus ticks in southern Slovakia. A part of the protein E gene was sequenced and compared with the prototype strain Neudorfl. Seventeen silent mutations and two amino acid changes (Ile-->Val, residue 167; Asn-->Thr, residue 366) were found. The nucleotide homology in the sequenced part of protein E gene of the strain 595 and the prototype strain Neudorfl is 98.6%. These findings indicate that the strain 595 is closely related to the strain Neudorfl.