TBP-1 protects the human oncosuppressor p14ARF from proteasomal degradation.

The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer. The mechanisms that regulate alternative reading frame (ARF) turnover have been obscure for long time, being ARF a relatively stable protein. Recently, it has been described that its degradation depends, at least in part, on the proteasome and that it can be subjected to N-terminal ubiquitination. We have previously reported that ARF protein levels are regulated by TBP-1 (Tat-Binding Protein 1), a multifunctional protein, component of the regulatory subunit of the proteasome, involved in different cellular processes. Here we demonstrate that the stabilization effect exerted by TBP-1 requires an intact N-terminal 39 amino acids in ARF and occurs independently from N-terminal ubiquitination of the protein. Furthermore, we observed that ARF can be degraded in vitro by the 20S proteasome, in the absence of ubiquitination and this effect can be counteracted by TBP-1. These observations seem relevant in the comprehension of the regulation of ARF metabolism as, among the plethora of cellular ARF's interactors already identified, only NPM/B23 and TBP-1 appear to be involved in the control of ARF intracellular levels.

Polyomavirus mutation that confers a cell-specific cis advantage for viral DNA replication.

The structural and biological properties of a polyomavirus mutant selected in Friend erythroleukemic cells were investigated. The growth efficiency of this mutant (PyFL78) was compared with that of the parental PyA2 strain by a growth competition assay in Friend erythroleukemic and 3T3 (or 3T6) cell lines. The results reveal that PyFL78 displays a cis-acting growth advantage over the PyA2 parental strain in Friend erythroleukemic cells but not in 3T3 or 3T6 cells. This cell-specific cis advantage is shown to be due to modifications within the polyomavirus noncoding regulatory region.

p14ARF interacts with the focal adhesion kinase and protects cells from anoikis.

The ARF protein functions as an important sensor of hyper-proliferative stimuli restricting cell proliferation through both p53-dependent and -independent pathways. Although to date the majority of studies on ARF have focused on its anti-proliferative role, few studies have addressed whether ARF may also have pro-survival functions. Here we show for the first time that during the process of adhesion and spreading ARF re-localizes to sites of active actin polymerization and to focal adhesion points where it interacts with the phosphorylated focal adhesion kinase. In line with its recruitment to focal adhesions, we observe that hampering ARF function in cancer cells leads to gross defects in cytoskeleton organization resulting in apoptosis through a mechanism dependent on the Death-Associated Protein Kinase. Our data uncover a novel function for p14ARF in protecting cells from anoikis that may reflect its role in anchorage independence, a hallmark of malignant tumor cells.

Suppression of Ras-mediated NIH3T3 transformation by p19ARF does not involve alterations of cell growth properties.

The INK4a gene, one of the most frequently disrupted loci in human cancer, encodes two unrelated proteins, p16INK4a and p19ARF, that both block cell proliferation. p16INK4a is a component of the Rb regulatory pathway, while p19ARF has been functionally related to p53. Moreover, p16INK4a is inactivated in many human tumors, while it has been very recently reported that p19ARF null mice develop tumors early in life. We show here that p19ARF is able to inhibit the formation of G418-resistant colonies when transfected into human and mouse cell lines expressing wild-type p53, regardless of p16 status. Moreover its amino terminal domain encoded by exon 1beta is still sufficient to obtain the same effect. We have analysed the ability of p19ARF to interfere with Ras-mediated cellular transformation in the NIH3T3 cell line. Cotransfection of p19ARF together with activated ras potently inhibited the formation of transformed foci in a dose-dependent manner. We have also isolated stable NIH3T3 transfectants expressing p19ARF and we have measured their growth properties as well as their efficiency of transformation by activated ras. Our results suggest that p19ARF can interfere with oncogene-mediated transformation, without significantly affecting NIH3T3 cell growth, at least at the levels of expression achieved in these experiments.

Characterization and genomic mapping of chimeric ERV9 endogenous retroviruses-host gene transcripts.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We performed Northern blot experiments using sequences from ERV9-LTR, and we observed a different pattern of expression in several different hemopoietic tumor cell lines. It is possible that by the result of a somatic integration event, or by virtue of their original dispersal in the genome, ERV9-LTRs may specifically induce the expression of different cellular sequences in different cell lineages. Here, we describe the identification and analysis of four chimeric cDNA clones isolated from the T-lymphoma Peer cell line, having a structure consistent with transcription initiation from an ERV9-LTR. All the cDNA clones represent transcripts derived from unique cellular sequences. We also report the genomic localization of these cDNA clones.

The transcription of B2 repeated sequences is regulated during the transition from quiescent to proliferative state in cultured rodent cells.

The RNA polymerase III-dependent transcription of B2 repeated sequences has been monitored during the transition from the quiescent to proliferative state in cultured rodent cells and after polyomavirus-induced transformation. The level of RNAs containing B2 sequences was found to be higher in both the proliferative state of normal cells and in polyomavirus-transformed cells. In both systems, nuclear run-off transcription assays indicated that high levels of B2 RNAs are due to an enhanced transcription rate. These results suggest the presence of a B2-specific RNA pol III transcription factor(s) whose activity is sensitive to cell cycle progression and oncogenic transformation.

Sequences both 5' and 3' to the transcription initiation site contribute to the ability of a mouse H-2 gene to respond to type I interferon.

To investigate the cis-acting DNA elements that are involved in the regulation of class I major histocompatibility complex genes by interferon, several promoter fragments of the H-2Kk gene were linked to the reporter chloramphenicol acetyl transferase (CAT) gene, and the CAT expression was analyzed in stable transfected cell lines. The functional activities of progressive deletions of the 5'-flanking region of the H-2Kk gene linked to the CAT gene have allowed us to define a discrete cis-acting DNA region necessary for interferon-mediated stimulation. Moreover, the H-2Kk gene transcribed by the nonregulated SV40 early promoter was also found to be under interferon regulation. Thus interferon enhancement of the H-2Kk gene expression appears to be mediated by two cis-acting elements, one located in the 5'-flanking region and the other by sequences downstream from the transcription initiation site.

rDNA magnification in D. melanogaster: state of rDNA copies following the first step.

D. melanogaster males of XYbb/O genetic constitution undergoing rDNA magnification were mated singly to XXbb+/O females, yielding XYbb/O male progeny, and to XNO- w sn bb+ females, yielding XYbb/XNO- females. The male and female offspring were scored for the bb+ phenotype. Results show that there is a higher percentage of bb+ flies in the XYbb/O male progeny than in XYbb/XNO- female progeny, in single crosses as well as in the combined data. rRNA/DNA hybridization experiments agree with this observation, by showing that the rDNA content in the progeny of premagnified flies was higher in the sons than in the daughters. These data indicate that the increase of ribosomal RNA genes is not due to a stable event such as an unequal mitotic sister exchange, whereas they do not contrast with the extracopy model.

Mutational analysis of the human endogenous ERV9 proviruses promoter region.

ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. To define all the elements required for the correct transcription activity of the ERV9 promoter and to establish a precise correlation between the elements important for basal transcription, we have systematically analyzed the in vivo and in vitro transcriptional activity of many different ERV9 promoter mutants, including a series of linker-scanning mutations across the promoter region. We report here that the ERV9 promoter contains two elements controlling the selection of the correct start sites, a TATA box and an Inr-like region; the concerted action of both elements is necessary for faithful transcription. Finally, using a series of GAL4 protein fusion constructs in cotransfection experiments, we demonstrated that various transcription factors can synergistically induce a high level of transcription when bound to an ERV9 DNA promoter.

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.

Different behaviour of Xbb+ and Xbb chromosomes in D. melanogaster with only one nucleolus organizer.

We have examined the rDNA content of male and female adult flies having only one nucleolus organizer (NO), using X chromosomes carrying wild or partially deleted bobbed loci (Xbb+/O, Xbb+/XNO- and Xbb/O, Xbb/XNO-). The results show that in Xbb+/O and Xbb+/XNO- flies, where only somatic gene compensation is supposed to occur, the rDNA increase, althought less pronounced than previously reported, is directly proportional to the number of rRNA genes initially present in the nucleolus organizer. In Xbb/O and in Xbb/Xbb/XNO- flies the rDNA increase is relatively much higher than that observed in flies carrying bb+ instead of bb. It is suggested that this may be due to rDNA premagnification and somatic gene compensation occurring simultaneously in the former flies.

Regulation of viral and cellular promoter activity by polyomavirus early proteins.

The chloramphenicol-acetyl-transferase (CAT) expression system has been utilized to study the ability of the polyomavirus (Py) early proteins, the 100K large T, the 55K middle T and 22K small T-antigens, to activate a variety of eukaryotic promoters (the SV40 early, the alpha 2(1) collagen, the rabbit beta-globin, the polyomavirus early and the H-2 class I) in both transient and stable expression assays. We have found that either the complete polyomavirus early region or a plasmid expressing only the 55K middle T-antigen are capable of stimulating the expression of all the promoter-CAT plasmids in transient co-transfection experiments in both NIH-3T3 and Rat-2 cells. Conversely, the Py early proteins do not stimulate the transcription of most of the promoter-CAT genes stably introduced in the cell chromosomes, with the exception of H-2 class I promoter, when stimulation of transcription has been observed upon infection with recombinant retrovirus encoding the Py middle T-antigen.

A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.

Characterization and genomic mapping of the ZNF80 locus: expression of this zinc-finger gene is driven by a solitary LTR of ERV9 endogenous retroviral family.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We describe here the structure of the ZNF80 cDNA clone putatively coding for a zinc-finger protein, whose 5' terminus starts from within an ERV9-LTR. Characterization of the single copy genomic locus indicates that a complete ERV9-LTR element is present upstream of the ZNF80 coding region and that this element acts as a functional promoter in both in vivo and in vitro experiments. A 2.6 kb long transcript is selectively expressed only in some hematopoietic cell lineages. Interestingly we mapped the ZNF80 locus to the 3q13.3 band, a region involved in karyotype rearrangements associated with myelocytic disorders. We have also analyzed the ZNF80 genomic organization in African green monkey and we show that this lower primate does not harbour an ERV9 element at this locus. Our findings strongly suggest that the expression of a zinc finger gene, which is highly conserved during evolution of primates, is regulated in humans by an LTR element of the ERV9 family.

Mobilization of an ERV9 human endogenous retroviral element during primate evolution.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We report here the structural organization in different primate species of a zinc-finger coding gene whose expression is driven in humans by a solitary ERV9-LTR promoter. Using a PCR strategy and library screening, we were able to trace the origin of the insertion event in the primate lineage and to evaluate the impact of this event on gene structure. Our findings indicate that the integration of the ERV9 element occurred after the split of orangutang from the great apes, but before the divergence of the gorilla lineage. These results suggest that ERV9 elements have been mobile within the primate lineages and may still be active in humans.

Structural and functional organization of the human endogenous retroviral ERV9 sequences.

The human genome contains a variety of genetic elements similar in structure to retroviruses and retrotransposons. We report here the structural and functional organization of a novel human endogenous retroviral family (ERV9). Three polyadenylated RNAs, 8, 2, and 1.5 kb long, are detected by Northern blot in undifferentiated embryonal carcinoma NT2/D1 cells. Upon genomic cloning of an expressed ERV9 locus, we demonstrated that the three polyadenylated RNAs are originated by a single ERV9 locus by alternative usage of splicing and polyadenylation signals. DNA sequence analysis of different ERV9 LTRs have revealed that they are heterogeneous in length and that the length variability is due to the number of tandemly repeated subelements present in both U3 and U5 regions; moreover, the ERV9 LTRs are capable to drive expression of a reporter gene in transient expression assays. Finally, analysis of the ERV9 5' transcription start site has allowed us to define the U3-R-U5 organization of the ERV9 LTR.

Isolation of cellular DNA sequences that allow expression of adjacent genes.

We have employed a strategy for the isolation and identification of cellular control (expression) sequences dependent on their ability to confer expression on a selectable gene devoid of its own expression sequences. The polyoma virus (Py) Hae II-BamHI DNA fragment, which comprises 84% of the intact viral DNA and contains the Py transforming region but lacks Py 5' expression sequences, was decreased markedly in its transformation of rat cells. Hae II-cleaved mouse cellular DNA was ligated to the Py Hae II-BamHI fragment. A transformed colony (H1) isolated after transfection of the ligated DNA onto rat cells was found to contain multiple inserts of Py DNA, most of which were biologically inactive. A transformed colony (H2) isolated after transfection of rat cells with total H1 DNA was found to contain a single insert of Py DNA. The H2 cells are highly tumorigenic and synthesize the three Py tumor antigens. Initiation of transcription of the Py early mRNAs in H2 cells occurs at the same Py nucleotides as in complete Py DNA. The viral and adjacent cellular DNA sequences were cloned from H2 cellular DNA. The transforming efficiency of the cloned Py transforming region and adjacent H2 cellular DNA was 20-40% of that of the viral DNA containing Py expression sequences. By BAL-31 deletion mapping it was observed that the first 58 base pairs of H2 cellular DNA were sufficient for the expression of the Py-transforming region. The sequence of the first 149 base pairs of the H2 cellular DNA was determined and does not show any striking similarities to upstream 5' sequences of a number of viral and host structural genes. Features of the H2 cellular sequence are discussed.

Activation of major histocompatibility complex class I mRNA containing an Alu-like repeat in polyoma virus-transformed rat cells.

Class I genes of the major histocompatibility complex (MHC) appear to be activated in mouse cells transformed by the DNA tumour virus simian virus 40 (SV40). Conversely, suppression of MHC class I genes has been reported in adenovirus-12-transformed baby kidney rat cells. We have now investigated the expression of genes encoded by the rat MHC locus in rat fibroblast cells transformed by polyoma virus (Py). Using a mouse genomic H-2 clone as a probe in Northern transfer hybridization analysis, we have observed a high level of expression of rat MHC class I messenger RNA in all the transformed rat cell lines analysed. The class I 1.6-kilobase (kb) mRNA activated in Py-transformed rat cells appears to contain an Alu-like type II repeat element, as the same 1.6-kb mRNA is detected using either the H-2 class I sequence or a repetitive Alu-like type II element as a probe. High levels of heterogeneous poly(A)+ transcripts of 0.5-0.8 kb are also observed in Py-transformed rat cells using probes containing an Alu-like type II repetitive element.

trans-activation of cellular and viral promoters by a transforming nonkaryophilic simian virus 40 large T antigen.

We used the chloramphenicol acetyl transferase (CAT) transient expression system to study the transactivating ability of a simian virus 40 (SV40) mutant that was unable to transport and localize large T antigen into the nucleus and which retained the competence to transform established cell lines. The effect of the SV40 wild type and the SV40 mutant for the large T antigen was tested in both mouse and simian cells on a series of plasmids in which the CAT gene was regulated by one of the following promoters: SV40 early and late, herpes simplex virus thymidine kinase, chicken alpha 2(I) collagen, mouse H-2Kk. Our results indicated that both the SV40 wild type and the cytoplasmic mutant for the large T antigen regulated transcription from any promoter tested, suggesting that the trans-activation by SV40 does not require the nuclear localization of the 100,000-molecular-weight large T-antigen protein.

Identification of new human repetitive sequences: characterization of the corresponding cDNAs and their expression in embryonal carcinoma cells.

We have identified new repeated interspersed DNA sequences by analysis of homologous RNA transcripts from a human teratocarcinoma cell line (NTERA-2 clone D1). The abundance of transcripts varies upon retinoic acid induced differentiation of NTERA-2/D1 cells, and it is highest when the cells display the embryonal carcinoma phenotype. The expression of these novel repeated sequences appears to be tissue specific as no detectable expression was found in various cell lines of different embryological derivation. Characterization of the RNA transcripts by analysis of recombinant cDNA clones indicated that transcripts of different genomic units are present in undifferentiated embryonal teratocarcinoma cells. Nucleotide sequencing of the cloned cDNAs reveals a complex structure composed by unique and tandemly repeated sub-elements.