The PDZ protein erbin modulates beta-catenin-dependent transcription.

Erbin is a member of the leucine-rich repeat and PDZ domain family that can regulate proliferation, differentiation and cell adhesion. As a binding partner of the receptor tyrosine kinase ErbB2, erbin targets this receptor to the basolateral membrane of polarized epithelial cells. In addition, erbin is known to inhibit the Ras-mediated activation of the mitogen-activated protein kinase pathway. Recently we identified the proto-oncoprotein beta-catenin as a ligand of the PDZ domain of erbin. Here we demonstrate that erbin acts as a negative regulator of the beta-catenin/T-cell-factor-dependent gene expression. In contrast, a mutant of erbin with a deletion of the N-terminal leucine-rich repeat allows the PDZ domain of erbin to increase the beta-catenin/T-cell-factor-dependent transcription. This mutant localizes to the nucleus and mimics a putative splice variant found in keratinocytes. Thus, erbin has the potential to act as an inhibitor as well as an activator of the beta-catenin-regulated gene expression.

Relationship between retroviral replication and RNA interference machineries.

Small interfering RNAs (siRNAs) associated with gene silencing are cellular defense mechanisms against invading viruses. The viruses fight back by suppressors or escape mechanisms. The retroviruses developed a unique escape mechanism by disguising as DNA proviruses. An evolutionary relationship between the siRNA machinery and the replication machinery of retroviruses is likely. The RNA cleavage enzymes PIWI and RNase H proteins are structurally related. This relationship can be extended from structure to function, since the retroviral reverse transcriptase (RT)/RNase H can also cause silencing of viral RNA by siRNA. Thus, both enzymes can cleave RNA-DNA hybrids and double-stranded RNA (dsRNA) with various efficiencies shown previously and here, demonstrating that their specificities are not absolute. Other similarities may exist, for example between PAZ and the RT and between RNA-binding proteins and the viral nucleocapsid protein. Dicer has some similarities with the viral integrase, since both specifically generate dinucleotide 3'-overhanging ends. We described previously the destruction of the human immunodeficiency virus (HIV) RNA by a DNA oligonucleotide ODN (oligodeoxynucleotide). Variants of the ODN indicated high length and sequence specificities, which is reminiscent of siRNA and designated here as "siDNA." Cleavage of the viral RNA in the presence of the ODN is caused by the retroviral RT/RNase H and cellular RNase H activities. Several siRNA-mediated antiviral defense mechanisms resemble the interferon system.

Prevention of angiogenesis by naked DNA IL-12 gene transfer: angioprevention by immunogene therapy.

IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-gamma and angiostatic CXCR3 chemokine ligands. Naked DNA intramuscular injection of an expression vector plasmid producing IL-12 resulted in significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant, inhibition. Control plasmid injection did not affect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell-depleted C57/bl and nude mice as well as in IFN-gamma(-/-) and CXCR3(-/-) knockout mice, indicating that NK- and/or T-cell-initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. Finally, IL-12 plasmid DNA gene transfer significantly prevented the growth and vascularization of highly angiogenic KS-Imm Kaposi's sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. These data suggest that a preventive gene therapy approach using antiangiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angioimmunoprevention.

The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain.

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state.

Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells.

The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of an in vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser(259), resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser(259) phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells.

Tumor regression induced by intratumoral injection of DNA coding for human interleukin 12 into melanoma metastases in gray horses.

Preclinical studies investigating new therapeutic principles against melanoma are presently being carried out in mouse models; however, these are not optimal. Here we describe a novel animal model using gray horses. These animals spontaneously develop metastatic melanoma that resembles human disease and is thus highly relevant for preclinical studies testing new immunotherapy protocols. We found that injection of plasmid DNA coding for the human cytokine interleukin 12 into established metastases induced significant regression in all 12 treated lesions in a total of 7 horses. Complete disappearance was observed in one treated lesion, with no recurrence after 6 months. No adverse events have been observed in any of the animals during and after treatment. These results demonstrate the effectiveness and safety of interleukin 12 encoding plasmid DNA therapy against established metastatic disease in a large animal model and serve as a basis for a clinical trial.

Synergistic effect of a combined DNA and peptide vaccine against gp100 in a malignant melanoma mouse model.

Vaccination against tumors relies on tumor-associated antigens, and has been quite successful with synthetic peptides used as immunogens. Gp100 is a human melanoma-associated antigen (hgp100) with a highly homologous mouse counterpart, pmel17/gp100 (mgp100), that is expressed in melanocytes and highly tumorigenic B16 melanoma cells. Since mgp100 is poorly immunogenic in mice, we used a xenoimmunization approach and vaccinated with the hgp100 immunogene. To that end, plasmid DNA encoding hgp100 was applied as a vaccine in combination with three synthetic peptides corresponding to putative cytotoxic T cell epitopes of hgp100. Immunization with DNA and peptide-pulsed spleen cells had a synergistic effect and provided significant protection against a challenge with poorly immunogenic B16-F0 malignant melanoma cells in the syngeneic C57BL/6 mouse model. Vaccination with either plasmid DNA or peptides alone delayed the onset of tumor formation, and reduced tumor growth 2-fold and 30-fold, respectively. However, while all animals vaccinated with DNA encoding hgp100 or with peptides eventually developed tumors, 30% of the animals treated with both vaccines remained tumor free and survived for the entire observation period of 150 days. Depletion of T cell subsets revealed that the protective effect observed after vaccination with plasmid DNA was mediated by CD4+ and CD8+ T cells, while protection following vaccination with DNA encoding hgp100 in combination with peptides appears to depend on CD4+ T cells only. Furthermore, we could also demonstrate a therapeutic effect of the combined DNA/peptide regime. A single treatment cycle consisting of injections of plasmid DNA and peptide-pulsed spleen cells led to a fourfold reduction in the growth rate of preexisting tumors. The data presented demonstrate that immunization with xenoantigens induces cross-species priming leading to an immunological response against the tumor-specific antigens.

Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation.

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.

Phase I clinical trial with HIV-1 gp160 plasmid vaccine in HIV-1-infected asymptomatic subjects.

The aim of the study was to investigate the safety of an HIV-1 gp160 plasmid vaccine. Four asymptomatic HIV-1-infected subjects with CD4+ lymphocyte counts >500/microl were injected with four times 400 microg of HIV-1 modified gp160 env and rev coding DNA vaccine at 0, 4, 10 and 28 weeks. Safety parameters, including autoimmune antibodies as well as CD4+/CD8+ cell counts and HIV-1 plasma concentrations, were monitored for 52 weeks after the first vaccine application. Follow-up data for more than 3 years are now available. The DNA vaccine proved to be safe and, specifically, did not induce anti-DNA autoimmune antibodies. Vaccination had no long-term effects on the CD4+/CD8+ lymphocyte counts, plasma HIV-1 RNA concentrations or disease progression. The present data supplement published data from Philadelphia, USA, where a dose-escalating study (30-300 microg) with the same HIV-1 DNA vaccine was performed.

Induction of putative tumor-suppressing genes in Rat-1 fibroblasts by oncogenic Raf-1 as evidenced by robot-assisted complex hybridization.

The growth factor receptor-dependent protein kinase Raf-1 is activated by GTP-bound Ras, thereby activating the mitogen-activated protein kinase pathway. To study the role of Raf in transformation we transduced Rat-1 cells with a tetracycline-regulatable retroviral vector encoding the constitutively active oncogenic C-terminal fragment of the human Raf-1 protein. Using subtractive hybridization of mRNAs from induced and noninduced cells and robot-assisted screening by complex hybridization, Raf-induced genes with various different characteristics of induction were investigated. Among the strongly induced genes were those involved in carcinogenesis such as metalloproteinases 3, 10 and 13, cathepsin L, ornithine decarboxylase, and putative tumor-suppressing genes such as monocyte chemoattracting protein 1, interferon-induced protein 10, a recently identified 2'-5' oligoadenylate synthetase-like protein, and plasminogen activator inhibitor type 2. Other components of the plasminogen activator system were not induced. Plasminogen activator inhibitor type 2 is a down-regulator of the proteolytic cascade consisting of various metalloproteinases, some of which are induced by a carboxy-terminal Raf mutant (RafCT). In conclusion, RafCT induces factors which act in a conflicting manner in respect of carcinogenesis, especially within the proteolytic system of the extracellular matrix.

The combined action of IL-15 and IL-12 gene transfer can induce tumor cell rejection without T and NK cell involvement.

The cooperative antitumor effects of IL-12 and IL-15 gene transfer were studied in the N592 MHC class I-negative small cell lung cancer cell line xenotransplanted in nude mice. N592 cells engineered to secrete IL-15 displayed a significantly reduced tumor growth kinetics, and a slightly reduced tumor take rate, while N592 engineered with IL-12 displayed only minor changes in their growth in nude mice. However, N592 cells producing both cytokines were completely rejected, and produced a potent local bystander effect, inducing rejection of coinjected wild-type tumor cells. N592/IL-12/IL-15 cells were completely and promptly rejected also in NK-depleted nude mice, while in granulocyte-depleted animals a slight delay in the rejection process was observed. Immunohistochemical analyses of the N592/IL-12/IL-15 tumor area in intact nude mice revealed the presence of infiltrating macrophages, granulocytes, and NK cells, and expression of inducible NO synthase and of secondary cytokines such as IL-1beta, TNF-alpha, and IFN-gamma, and at higher levels GM-CSF, macrophage-inflammatory protein-2, and monocyte chemoattractant protein-1. In NK cell-depleted nude mice, numerous macrophages and granulocytes infiltrated the tumor, and a strong expression of macrophage-inflammatory protein-2 and inducible NO synthase was also observed. Finally, macrophages cocultured with N592/IL-12/IL-15 produced NO in vitro, and inhibited tumor cell growth, further suggesting their role as effector cells in this model.

Inhibition of human hematopoietic tumor formation by targeting a repressor Myb-KRAB to DNA.

In many hematopoietic malignancies, c-Myb, a nuclear transcription factor of hematopoietic cells, is an activated oncogene. To achieve a specific inhibition of hematopoietic tumor growth, an inducible fusion protein consisting of the Myb DNA binding domain (DBD) and the active repressor domain KRAB, the Krüppel-associated box of the developmental zinc-finger protein KOX-1, was generated. The MybDBD-KRAB fusion protein is a potent repressor of Myb-induced gene expression from Myb-responsive reporter genes containing several Myb binding sites. MybDBD-KRAB expressed in the human hematopoietic promyelocytic cell line HL60 significantly reduces cell proliferation by inducing apoptosis. Expression of MybDBD-KRAB in subcutaneously injected HL60 cells leads to inhibition of tumor formation in nude mice. The MybDBD-KRAB effect is specific to cell lines expressing c-Myb. It is conceivable to fuse the KRAB domain to other DBDs of oncogenic transcription factors and target them to their respective DNA response elements to selectively drive tumor cells into apoptosis.

Retroviral gene transfer of dominant negative raf-1 mutants suppresses ha-ras-induced transformation and delays tumor formation.

Activating mutants of ras are among the most frequently found genetic alterations in human cancers. Therefore, Ras appears to be an attractive target for therapeutic intervention using gene transfer. The protein kinase Raf-1 acts as a direct downstream effector of Ras and is involved in Ras-induced cellular transformation. Using the NIH3T3 fibroblast-derived tumor cell line PEJ, which expresses oncogenic Ha-rasG12V, we analyzed whether dominant negative mutants of Raf-1 can inhibit Ras-mediated transformation. Retroviral gene transfer was used to stably transduce PEJ cells with three different dominant negative mutants of Raf-1. This resulted in reversion of the transformed phenotype in vitro as evidenced by an increase in contact inhibition and reduced anchorage-independent growth. However, tumor formation in nude mice was significantly delayed only by one of these mutants. Therefore, dominant negative mutants of the oncoprotein Myc, which is known to synergize with Raf-1 in tumor formation, were transduced into PEJ cells expressing a dominant negative Raf mutant. This leads to killing of the cells. These results indicate that although interference with Ras-induced transformation using dominant negative mutants of Raf is feasible and effective in vitro using retroviral vectors, an additional block (e.g., that of Myc) is necessary to kill PEJ cells. These results also indicate that interference with Ras-dependent signaling is not sufficient for inhibition of tumor formation of PEJ cells in vivo.

Tetracycline-induced expression of an anti-c-Myb single-chain antibody and its inhibitory effect on proliferation of the human leukemia cell line K562.

Ablation of c-Myb function might be an effective approach for the therapy of chronic myelogenous leukemia or other c-myb-dependent malignancies. To this end, we have previously used an intracellular anti-c-Myb single-chain antibody (sFv) to achieve the functional knockout of the c-Myb oncoprotein. In this study, we have employed a tetracycline-inducible system to control the expression of the sFv. A nuclear-localizing form of an anti-c-Myb sFv was cloned into a tet-regulated plasmid vector. Using a transient expression system in COS-1 cells, we observed that doxycycline (Dox) induced expression of the sFv in a dose-dependent manner, and that the sFv was localized mainly in the nucleus. The Dox-induced anti-c-Myb sFv also inhibited the transactivating activity of c-Myb in a dose-dependent manner. We subsequently confirmed the Dox-induced expression of the sFv in the leukemia cell line K562. Proliferation of the target leukemia cells was also inhibited. These results suggest that the anti-c-Myb sFv may represent a viable method for gene therapy of c-myb-dependent hematopoietic malignancies.

Induction of long-lasting cytokine effect by injection of IL-12 encoding plasmid DNA.

We have recently demonstrated that DNA coding for both subunits of the murine IL-12 heterodimer exhibits a strong antimetastatic effect against B16-melanoma in C57BL/6 mice and after intratumoral injection tumor regression. Here we show that the antimetastatic effect can be detected when the DNA is injected intramuscularly 30 days before tumor cell challenge. A long-term IL-12 expression was measured for up to 50 days in the serum with a peak at day 20 amounting to about 10 ng/mL in C57BL/6 mice. CpG oligodeoxynucleotides also induce IL-12 expression, however, only for a few hours. IL-12 DNA administration induces long-lasting systemic IFN -gamma production, whereas IL-4 and TNF-alpha levels remained undetectable. NK cell-depleted mice showed a strong but reduced expression of murine IL-12. Expression of DNA encoding human instead of murine IL-12 resulted in a significantly lower and transient expression, indicating that not plasmid-derived IL-12 production alone but the immune system of the host contributes to the long- lasting antimetastatic effect. It may be attributable to an autocrine feedback mechanism maintaining murine IL- 12 expression, whereby several cell populations including NK cells are involved.

Immune modulation in cancer using DNA inoculation--antitumour effect of interleukin-12.

DNA vaccines hold great promise for the prevention and therapy of infectious diseases and cancer. Furthermore, DNA has a high potential value not only for vaccination but also for therapy. Gene products which exhibit high potential biological potency, even at low levels of expression, are the most promising candidates. We have recently demonstrated that intramuscular injection of plasmid DNA coding for IL-12 abolishes the establishment of pulmonary metastases of 816F10 melanoma cells in a syngeneic mouse model. Based on these findings, we have evaluated the antitumour effect of IL-12 DNA therapy in a tumour model. Intramuscular injection of a murine IL-12-encoding DNA plasmid resulted in a pronounced reduction of tumour growth using preestablished syngeneic tumours in C57/BL6 mice. This antitumour effect correlated with a long-lasting expression of cytokines, which manifested itself as high levels of IL-12 in the serum 12 days after DNA treatment. The absence of significant toxic side effects could represent a significant advantage of IL-12 DNA therapy.

Immunization with Naked DNA Coexpressing Antigen and Cytokine via IRES.

Inoculation of plasmid DNA vectors encoding immunogenic proteins induce humoral as well as cell-mediated immune responses. Protection against challenge with pathogens has provided protective immunity in several instances in animal models. (1,2). DNA vaccines allow the simultaneous expression of antigens and immune-stimulatory cytokines via an internal ribosomal entry site (IRES). Here we describe the construction of a DNA vaccine against malignant melanomas using: (i) the tumor-associated antigen gp100 (or pmel17), known to be over-expressed in many malignant melanomas (3,4), and (ii) the granulocyte macrophage stimulating factor (GM-CSF) which has been shown to have a stimulatory effect on humoral and cellular immune responses (5).

Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt.

Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways were shown to exert opposing effects on muscle cell hypertrophy. Furthermore, the PI3K-Akt pathway was shown to inhibit the Raf-MEK-ERK pathway; this cross-regulation depended on the differentiation state of the cell: Akt activation inhibited the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlated with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.

Phosphorylation and regulation of Raf by Akt (protein kinase B).

Activation of the protein kinase Raf can lead to opposing cellular responses such as proliferation, growth arrest, apoptosis, or differentiation. Akt (protein kinase B), a member of a different signaling pathway that also regulates these responses, interacted with Raf and phosphorylated this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt.

Microencapsulation of DNA using poly(DL-lactide-co-glycolide): stability issues and release characteristics.

The design of DNA vaccination delivery systems for the targeting of professional antigen presenting cells could be an interesting approach to elicit cytotoxic T-cell responses to fight viral infections and in cancer therapy. Stability studies with linear high and low molecular DNA and supercoiled plasmid DNA were performed in order to check their ability to withstand stress conditions applied during formulation processes. DNA was tested for integrity by the PicoGreen assay and transfectivity was assessed in cell culture transfection experiments. Double-stranded DNA is extremely stable under physiological conditions in vitro but is rapidly degraded under acidic conditions and high shear forces. Thereby, different stress factors resulted in distinct degradation patterns such as fragmentation and strand separation possibly followed by further decomposition of single-stranded DNA. DNA containing PLGA microparticles as a potential delivery system was prepared by spray-drying. Encapsulation efficiency, DNA stability and burst release varied significantly depending on the different parameters explored in this study. The microencapsulation process was altered to achieve maximal stability of encapsulated DNA by reducing exposure to shear forces and by the addition of NaHCO(3) which acts as a buffering agent and furthermore stabilizes dsDNA against mechanical degradation. Stability of DNA is maintained during the burst release phase, but massive degradation occurred during the second release phase possibly due to acidic catalyzed decomposition. In summary, we feel that microencapsulation of DNA vaccines by spray-drying offers manifold possibilities to design suitable delivery systems in terms of optimizing phagocytosis by APCs and maintaining stability of DNA in phagosomes.