5' phospholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3, 4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G(1) in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27(KIP1). Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS) oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.

Evidence of a role for Ki-Ras in the stimulated proliferation of renal fibroblasts.

Progressive renal fibrosis is driven by a range of cytokines that act via membrane receptors and intracellular signaling cascades to evoke gene transcription events and related responses. The Ras family of GTPases has been implicated in many of these signaling cascades in model systems such as 3T3 fibroblasts. However, the roleof the specific Ras isoforms Ki, Ha, and N in the stimulation of renal fibroblasts has not been defined. In this study, Ras has been inhibited in primate renal fibroblasts (vero cells) using specific phosphorothioate oligodeoxynucleotides (oligos) targeting the three isoforms. Lipofectin transfection with 200 to 400 nM Ki-Ras oligo inhibited the epidermal growth factor- and fibroblast growth factor-stimulated proliferation of vero cells by 25 to 35% with a lesser effect on serum-stimulated growth. Oligos against Ha-Ras and N-Ras were inactive with respect to control oligo. Total cellular Ras protein (estimated by Western blotting) was reduced by 60 to 90% 24 h after transfection with Ki-Ras oligo. N-Ras, Ha-Ras, and control oligos were inactive. Total Ras synthesis over 4 h measured using [35S]-cys/met pulse chase was reduced by approximately 70% by Ki-Ras oligo and not altered by other oligos. The fractional prenylation of Ras was quantified from the discrete bands on polyacrylamide gel electrophoresis and was increased by the Ki-Ras oligo alone. These data demonstrate that these renal fibroblasts predominantly express the Ki isoform of Ras and that this GTPase plays a role in the stimulated proliferation of these cells. Ras GTPases may be a target for the inhibition of processes leading to renal fibrosis.

Application of antisense oligonucleotides for gene functionalization and target validation.

The Human Genome Project (complete sequencing of the human genome) will be complete soon and the information made available to the biomedical community. Although the project is not yet complete, it has dramatically changed the practice of biomedical sciences. With enormous amounts of information available from sequencing efforts, increasing demands are being put on researchers to quickly determine the biochemical function of novel molecular targets and to validate them as appropriate for drug discovery endeavors. Antisense oligonucleotides are an ideal technology for gene functionalization and target validation. They are an efficient methodology for gene functionalization and target validation and are a proven technology. Antisense technology can answer questions with a high degree of precision and it is a versatile technology. In this review the use of antisense oligonucleotides as a research tool for gene functionalization and target validation is discussed.

Interleukin-1 beta and reactive oxygen species mediate activation of c-Jun NH2-terminal kinases, in human epithelial cells, by two independent pathways.

The c-Jun N terminal kinases (JNKs) are members of the mitogen activated protein kinases family, which have been shown to be preferentially activated either by cytokines or stress stimuli. In this study we identify a selective and potent antisense oligonucleotide to RhoA (ISIS 17131) and investigate its effect on JNK activation induced by IL-1beta and H2O2 in A549 cells. The RhoA antisense oligonucleotide was able to inhibit JNK activation when A549 cells were stimulated by H2O2, but did not have any effect on IL-1beta induced JNK activation. Consistent with the idea that the phosphatidylinositol 3-kinase (PI 3-kinase) activates the small G protein exchange factors, H2O2 activated the PI 3-kinase. Additionally, Wortmannin, a potent inhibitor of the PI 3-kinase and phospholipase A2 (PLA2), and AACOCF3, also a PLA2 inhibitor, were able to inhibit JNK activation induced by H2O2, but they had no effect on JNK activation when stimulated by IL-1beta. These results suggest that, in A549, IL-1beta and H2O2 induce JNK activation by two independent pathways.

A novel drug screening assay for papillomavirus specific antiviral activity.

Discovery and development of human papillomavirus (HPV) specific antiviral agents have been hampered by the lack of an in vitro assay permissive to HPV replication. An experimental assay system for monitoring HPV-11 DNA replication has been optimized for use as a papillomavirus antiviral drug screening tool. Cloned HPV DNA was introduced into SCC-4 cells by electroporation and viral DNA replication monitored by Southern blot. Kinetic studies demonstrated an increased HPV genome copy number with time. Viral DNA replicated as episomal, unit length genome and remained episomal after multiple passages. These data suggested the basis for an in vitro replication assay for evaluating the antiviral activity of potential chemotherapeutic agents directly on HPV. This model was used to investigate antiviral activities of current anti-HPV therapies such as 5-fluorouracil (5-FU) and alpha-interferon (alpha-IFN) and potential therapies such as sodium butyrate, 5-bromo-20-deoxyuridine (BrdU) and antisense oligonucleotides. HPV- 11 replication is significantly inhibited by BrdU and sodium butyrate; however 5-FU and alpha-IFN did not give consistent dose response results. Finally, ISIS 2105, a 20-mer phosphorothioate antisense oligonucleotide, which targets HPV-11 E2 gene product, showed potent antiviral activity in this assay with an IC50 of approximately 70 nM.

Multiplicity of uses of monoclonal antibodies that define papillomavirus linear immunodominant epitopes.

During the last 10 yr, we have derived monoclonal antibodies from animals immunized with denatured bovine papillomaviruses type 1 major capsid (L1) protein, mapped their corresponding immunodominant epitopes to within a single amino acid (aa), and compared the reactivity of authentic L1 proteins to the predicted response by collinear analysis of the aa sequences of the same and other papillomaviruses (PVs). The data obtained from this approach has provided us with new insights into the sensitivity and specificity of the antibody response to viral proteins. We have included here some observations and conclusions that appear to be generic for the immune response, some of which might have applications for working with linear epitopes in other experimental systems.

Characterization of human papillomavirus-11 mRNAs expressed in the context of autonomously replicating viral genomes.

We have previously adapted an experimental system which supports autonomous replication of human papillomavirus-11 (HPV-11) genomic DNA in a human squamous carcinoma cell line (SCC-4) following electroporation of linearized viral DNA. This system allows evaluation of HPV-11 transcription in the context of replicating viral genomes. RNA was isolated from cells following transfection, and first-strand cDNA was produced by reverse transcription using HPV-11-specific primers. The cDNAs were amplified using the polymerase chain reaction, and resulting DNA products were cloned and sequenced. Transcripts were identified that utilize the same splice donor and acceptor sites as transcripts characterized previously from human lesions. Three previously unreported splice junctions that employ novel combinations of those splice sites were also identified. We also detected these newly identified transcripts in laryngeal papillomas. A modified version of the 5' rapid amplification of cDNA ends method was used to identify transcriptional start sites of several of the HPV-11 transcripts. The family of mRNAs characterized in this replication system have the potential to encode all of the major HPV-11 E-region proteins described to date. The data support the utility of this system as an experimental model for examining mechanisms of viral replication and transcription.

n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses.

Small DNA viruses are dependent on the interaction of early proteins (such as large T antigen) with host p53 and Rb to bring about the G1-to-S cell cycle transition. The large DNA viruses are less dependent on host regulatory genes since additional early viral proteins (such as viral DNA polymerase, DNA metabolic enzymes, and other replication proteins) are involved in DNA synthesis. A highly conserved domain of large T antigen (similar to the p53-binding region) exclusively identifies papovavirus, parvovirus, and papillomaviruses from all other larger DNA viruses and implies a conserved interaction with host regulatory genes. In this report, we show that 3 to 6 mM butyrate, a general cell cycle blocker implicated in inhibition of the G1-to-S transition, inhibits DNA replication of polyomavirus and human papillomavirus type 11 but not the replication of larger DNA viruses such as adenovirus types 2 and 5, herpes simplex virus type 1, Epstein-Barr virus, and cytomegalovirus, which all bypass the butyrate-mediated cell cycle block. This butyrate effect on polyomavirus replication is not cell type specific, nor does it depend on the p53 or Rb gene, as inhibition was seen in fibroblasts with intact or homozygous deleted p53 or Rb, 3T6 cells, keratinocytes, C2C12 myoblasts, and 3T3-L1 adipocytes. In addition, butyrate did not inhibit expression of polyomavirus T antigen. The antiviral effect of butyrate involves a form of imprinted state, since pretreatment of cells with 3 mM butyrate inhibits human papillomavirus type 11 DNA replication for at least 96 h after its removal. Butyrate, therefore, serves as a molecular tool in dissecting the life cycle of smaller DNA viruses from that of the larger DNA viruses in relation to the cell cycle.

Effects of human papillomavirus type 18-specific antisense oligonucleotides on the transformed phenotype of human carcinoma cell lines.

DNA of human papillomavirus type 18 is present in several human cancer cell lines that were derived from oral or cervical tumors, and it is known that several features of the transformed phenotype can be inhibited by expression of antisense RNA to human papillomavirus (HPV). The present study was performed to find out whether antisense oligonucleotides were also inhibitory. Synthetic oligonucleotides were made that were complementary to regions of the start codons of the E6 and E7 genes of HPV-18. These were added to cultures of the oral cancer cell line 1483 and the cervical cancer cell line C4-1, each of which contain DNA of HPV-18. As controls we used the oral cancer cell line 183 and the monkey kidney cell line Vero, which do not contain HPV. Anti-E6 and anti-E7 oligonucleotides, in concentrations between 1 and 5 microM, significantly inhibited the growth of the 1483 and C4-1 cells, but not the 183 or Vero cell lines. Treatment of the 1483 cells with a combination of 2.5 microM of each of the antisense oligonucleotides was a more effective inhibitor than 5 microM of either one used alone. Antisense oligonucleotides had no effect on the ability of 1483 cells to form foci in soft agar, nor on their plating efficiency or serum requirements. Microscopic examination of 1483 cells showed that the antisense E7 oligonucleotide produced cell-rounding, detachment from the surface of the culture flask, and cell death, while the antisense E6 oligonucleotide had none of these effects. Random-sequence oligonucleotides had no effects of any type on any cells that were growing in culture. However, if random-sequence oligonucleotides were added to cells at the time they were passed to a new culture vessel, they produced severe nonspecific toxic effects. These results show that the use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behavior of human cancer cells that contain DNA of HPV-18.

In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts.

Papillomaviruses induce benign proliferative lesions, such as genital warts, in humans. The E2 gene product is thought to play a major role in the regulation of viral transcription and DNA replication and may represent a rational target for an antisense oligonucleotide drug action. Phosphorothioate oligonucleotides complementary to E2 mRNAs were synthesized and tested in a series of in vitro bovine papillomavirus (BPV) and human papillomavirus (HPV) models for the ability to inhibit E2 transactivation and virus-induced focus formation. The most active BPV-specific compounds were complementary to the mRNA cap region (ISIS 1751), the translation initiation region for the full-length E2 transactivator (ISIS 1753), and the translation initiation region for the E2 transrepressor mRNA (ISIS 1755). ISIS 1751 and ISIS 1753 were found to reduce E2-dependent transactivation and viral focus formation in a sequence-specific and concentration-dependent manner. ISIS 1755 increased E2 transactivation in a dose-dependent manner but had no effect on focus formation. Oligonucleotides with a chain length of 20 residues had optimal activity in the E2 transactivation assay. On the basis of the above observations, ISIS 2105, a 20-residue phosphorothioate oligonucleotide targeted to the translation initiation of both HPV type 6 (HPV-6) and HPV-11 E2 mRNA, was designed and shown to inhibit E2-dependent transactivation by HPV-11 E2 expressed from a surrogate promoter. These observations support the rationale of E2 as a target for antiviral therapy against papillomavirus infections and specifically identify ISIS 2105 as a candidate antisense oligonucleotide for the treatment of genital warts induced by HPV-6 and HPV-11.

Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction.

The structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1) were determined at 2.5 nm resolution by cryoelectron microscopy and three dimensional image reconstruction techniques. As expected, the reconstructions showed that both viruses consist of a T = 7 icosahedral capsid (approximately 60 nm in diameter) which surrounds a nucleohistone core. The capsid morphologies of the two viruses are nearly indistinguishable. Each capsid consists of a shell layer (approximately 2 nm thick) of nearly continuous density from which capsomers project radially to a maximum height of approximately 5.8 nm. The five-coordinate (pentavalent) and six-coordinate (hexavalent) capsomers both exhibit distinct five-fold axial symmetry as was observed for SV40 and polyoma viruses. Thus, both genera (papilloma and polyoma) of the papovavirus family have now been shown to have the characteristic "all-pentamer" capsid construction. BPV-1 and HPV-1 capsomers consist of a thick (8.6 nm diameter) trunk that broadens distally to form a regular five-pointed, star-shaped head, and proximally to create the shell layer where capsomers associate. A cylindrical channel (approximately 2.8 nm diameter) extends along the axis of each capsomer from the interior of the virus to a point approximately half way to the capsomer surface. Computationally sectioned views of individual capsomers displayed at decreasing radii show that each of the five capsomer subunits (in both pentavalent and hexavalent capsomers) makes a pronounced (30 degrees) left-handed twist just above the outer surface of the capsid shell. Similar views of the reconstructions also clarify the morphology of intercapsomer contacts. For example, they show how hexavalent capsomers coordinate six neighboring capsomers despite the fact that they contain only five subunits. The system of intercapsomer contacts is indistinguishable in BPV-1 and HPV-1, but quite different from that reported for polyoma virus capsids assembled in vitro from the major capsid protein, VP1 (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea. 1989. Biophys. J. 56:887-900). Thus, because both polyoma and papilloma viruses have all-pentamer capsids, it appears that intracapsomer subunit-subunit interactions which stabilize pentameric capsomers are better preserved evolutionarily than those involved in capsomer-capsomer contacts.

Identification of linear epitopes of the BPV-1 L1 protein recognized by sera of infected or immunized animals.

Sera from cattle that had been inoculated with BPV-1 virions or with recombinant L1 proteins and serum from a rabbit that had been immunized with SDS-denatured virions were evaluated for their reactivity with 466 overlapping synthetic peptides corresponding to 95% of the BPV-1 L1 protein. The late serological response of cattle to both intact virions and recombinant L1 proteins exhibited a similar profile of reactivity with approximately 70% (7 of 10) of L1 antigenic sites. However, the L1 serological response of the rabbit to SDS-denatured virions exhibited a significant difference from bovine serum antibodies in the profile of epitopes recognized, including a relative lack of response to major bovine epitopes located between L1 amino acids (AAs) 300-400. Importantly, only the sera from animals inoculated/immunized with intact virions was capable of neutralizing BPV-1 infectivity of murine C127 cells, suggesting that nonlinear epitopes are important for papillomavirus neutralization.