Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Tumor necrosis factor (TNF)-mediated kinase cascades: Bifurcation of Nuclear Factor-κB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2

TNF-induced activation of the transcription factor NF-κB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-κB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-κB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved “WKI” motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-κB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-κB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-κB and JNK, respectively.

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Direct interaction of Gβγ with a C-terminal Gβγ-binding domain of the Ca2+ channel α1 subunit is responsible for channel inhibition by G protein-coupled receptors

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its βγ dimer (Gβγ). We report below the existence of two Gβγ-binding sites on the A-, B-, and E-type α1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gβγ-binding regions also bind the Ca2+ channel β subunit (CCβ), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in α1E of loop 1 with that of the G protein-insensitive and Gβγ-binding-negative loop 1 of α1C did not abolish inhibition by G proteins, but the exchange of the α1E C terminus with that of α1C did. This and properties of α1E C-terminal truncations indicated that the Gβγ-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gβγ to this site was inhibited by an α1-binding domain of CCβ, thus providing an explanation for the functional antagonism existing between CCβ and G protein inhibition. The data do not support proposals that Gβγ inhibits α1 function by interacting with the site located in the loop I–II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Cloning of the cDNA for the TATA-binding protein-associated factorII170 subunit of transcription factor B-TFIID reveals homology to global transcription regulators in yeast and Drosophila

The human transcription factor B-TFIID is comprised of TATA-binding protein (TBP) in complex with one TBP-associated factor (TAF) of 170 kDa. We report the isolation of the cDNA for TAFII170. By cofractionation and coprecipitation experiments, we show that the protein encoded by the cDNA encodes the TAF subunit of B-TFIID. Recombinant TAFII170 has (d)ATPase activity. Inspection of its primary structure reveals a striking homology with genes of other organisms, yeast MOT1, and Drosophila moira, which belongs to the Trithorax group. Both homologs were isolated in genetic screens as global regulators of pol II transcription. This supports our classification of B-TFIID as a pol II transcription factor and suggests that specific TBP–TAF complexes perform distinct functions during development.

Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells

Cytochrome b-type NAD(P)H oxidoreductases are involved in many physiological processes, including iron uptake in yeast, the respiratory burst, and perhaps oxygen sensing in mammals. We have identified a cytosolic cytochrome b-type NAD(P)H oxidoreductase in mammals, a flavohemoprotein (b5+b5R) containing cytochrome b5 (b5) and b5 reductase (b5R) domains. A genetic approach, using blast searches against dbest for FAD-, NAD(P)H-binding sequences followed by reverse transcription–PCR, was used to clone the complete cDNA sequence of human b5+b5R from the hepatoma cell line Hep 3B. Compared with the classical single-domain b5 and b5R proteins localized on endoplasmic reticulum membrane, b5+b5R also has binding motifs for heme, FAD, and NAD(P)H prosthetic groups but no membrane anchor. The human b5+b5R transcript was expressed at similar levels in all tissues and cell lines that were tested. The two functional domains b5* and b5R* are linked by an approximately 100-aa-long hinge bearing no sequence homology to any known proteins. When human b5+b5R was expressed as c-myc adduct in COS-7 cells, confocal microscopy revealed a cytosolic localization at the perinuclear space. The recombinant b5+b5R protein can be reduced by NAD(P)H, generating spectrum typical of reduced cytochrome b with alpha, beta, and Soret peaks at 557, 527, and 425 nm, respectively. Human b5+b5R flavohemoprotein is a NAD(P)H oxidoreductase, demonstrated by superoxide production in the presence of air and excess NAD(P)H and by cytochrome c reduction in vitro. The properties of this protein make it a plausible candidate oxygen sensor.

Role of Syk in B-cell development and antigen-receptor signaling

Antigen receptors (BCRs) on developing B lymphocytes play two opposing roles—promoting survival of cells that may later bind a foreign antigen and inhibiting survival of cells that bind too strongly to self-antigens. It is not known how these opposing outcomes are signaled by BCRs on immature B cells. Here we analyze the effect of a null mutation in the Syk tyrosine kinase on maturing B cells displaying a transgene-encoded BCR that binds hen egg lysozyme (HEL). In the absence of HEL antigen, HEL-specific BCRs are expressed normally on the surface of Syk-deficient immature B-lineage cells, but this fails to promote maturation beyond the earliest stages of B-lineage commitment. Binding of HEL antigen, nevertheless, triggers phosphorylation of CD79α/β BCR subunits and modulation of receptors from the surface in Syk-deficient cells, but it cannot induce an intracellular calcium response. Continuous binding of low- or high-avidity forms of HEL, expressed as self-antigens, fails to restore the signal needed for maturation. Compared with the effects in the same system of null mutations in other BCR signaling elements, such as CD45 and Lyn kinase, these results indicate that Syk is essential for transmitting a signal that initiates the program of B-lymphocyte maturation.

The transcriptional program of a human B cell line in response to Myc

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

Targeted disruption of the arylsulfatase B gene results in mice resembling the phenotype of mucopolysaccharidosis VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase B (ASB), which is involved in degradation of dermatan sulfate and chondroitin 4-sulfate. A MPS VI mouse model was generated by targeted disruption of the ASB gene. Homozygous mutant animals exhibit ASB enzyme deficiency and elevated urinary secretion of dermatan sulfate. They develop progressive symptoms resembling those of MPS VI in humans. Around 4 weeks of age facial dysmorphia becomes overt, long bones are shortened, and pelvic and costal abnormalities are observed. Major alterations in bone formation with perturbed cartilaginous tissues in newborns and widened, perturbed, and persisting growth plates in adult animals are seen. All major parenchymal organs show storage of glycosaminoglycans preferentially in interstitial cells and macrophages. Affected mice are fertile and mortality is not elevated up to 15 months of age. This mouse model will be a valuable tool for studying pathogenesis of MPS VI and may help to evaluate therapeutical approaches for lysosomal storage diseases.

In vitro reconstitution of human replication factor C from its five subunits.

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.

A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137). To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion of the nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.

Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.