Replication-defective Retrovirus Research Articles

The murine-reduced folate carrier gene can act as a selectable marker and a suicide gene in hematopoietic cells in vivo.

Increased expression of the reduced folate carrier confers sensitivity to the antifolate drug methotrexate because it results in increased cellular uptake of this drug, and increased resistance to trimetrexate, a lipid-soluble antifolate drug, because it enables cells to take up exogenous folates that rescue cells from antifolate cytotoxicity. We therefore hypothesized that the reduced folate carrier could act as a suicide gene after treatment with methotrexate and as a selectable marker after exposure to trimetrexate. To test this hypothesis, we constructed replication-defective retroviruses containing the murine-reduced folate carrier (mRFC). Murine bone marrow cells transduced with the mRFC-containing retrovirus showed increased sensitivity to methotrexate and increased resistance to trimetrexate compared to empty vector-transduced controls in colony forming assays. Furthermore, colonies surviving trimetrexate and methotrexate treatment showed an enrichment of the mRFC gene after exposure to trimetrexate and a decrease after exposure to methotrexate. Lethally irradiated mice transplanted with bone marrow cells transduced with the mRFC-retrovirus and treated with the antifolate drugs after hematopoietic recovery demonstrated a relative increase in the number of cells containing the mRFC transgene after trimetrexate treatment and a decrease after methotrexate treatment. Therefore, these studies demonstrate the potential of the reduced folate carrier gene to play a dual role in gene therapy applications.

Astrocyte Delivery of Glial Cell Line-Derived Neurotrophic Factor in a Mouse Model of Parkinson's Disease

Primary astrocytes were genetically modified ex vivo to express recombinant glial cell line-derived neurotrophic factor (GDNF) and subsequently were tested for their ability to provide neuroprotection to dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) mouse model of Parkinson's disease. A replication-defective retrovirus was constructed, which contained the rat GDNF sequence and a sequence encoding a β-galactosidase (β-gal)/neomycin phosphotransferase fusion protein, linked via an internal ribosomal entry site. Murine astrocytes transduced with this vector secreted GDNF into the culture media at the rate of 115 ± 34 pg/24 h/105 cells and expressed cytoplasmic β-gal, whereas control nontransduced astrocytes were negative for GDNF production and cytoplasmic β-gal expression. Mice that received implants of GDNF-producing astrocytes into the striatum or nigra displayed elevated levels of GDNF compared to mice that received control nontransduced astrocytes. In addition, tissue content of GDNF was increased bilaterally and in brain regions both proximal and distal to the graft, even though astrocyte migration away from the graft site did not occur. Importantly, GDNF-producing astrocytes provided marked neuroprotection of nigral dopaminergic perikarya, and partial protection of striatal dopaminergic fibers, when implanted into the midbrain 6 days prior to a retrograde 6-OHDA lesion, as assessed by tyrosine hydroxylase immunohistochemistry. Similarly, GDNF-producing astrocytes prevented the acquisition of amphetamine-induced rotational behavior in 6-OHDA-treated mice and completely prevented dopamine depletion within the substantia nigra, as assessed by high-performance liquid chromatography. These results indicate that continuous exposure to low levels of GDNF provided by transgenic astrocytes provides marked neuroprotection of nigral dopaminergic neurons.

Induction of Tubulogenesis in Telomerase-Immortalized Human Microvascular Endothelial Cells by Glioblastoma Cells

To facilitate the study of human endothelial cells we have used a replication defective retrovirus encoding the catalytic subunit of telomerase (hTERT) to derive populations of telomerase-immortalized human microvascular endothelial (TIME) cells. Whereas parental HMVECs became senescent on average within 35–45 population doublings (PDs), TIME cells have continued to proliferate for at least 200 PDs. TIME cells express readily detectable telomerase activity but display only a modest increase in telomere length. Karyotypic analysis reveals the cells to have a normal complement of human chromosomes with no evidence of gross genetic abnormalities. Furthermore, TIME cells retain many of the characteristics of the primary endothelial cells from which they were derived. For example, they express a panel of characteristic endothelial cell surface marker proteins such as CD31/PECAM-1 and αvβ3-integrin. In addition, TIME cells express receptors for low-density lipoprotein (LDL) receptor as they are competent for receptor-mediated endocytosis of fluorescent acetylated LDL. Importantly, when plated on matrigel, TIME cells undergo tubule formation. Moreover, when cocultured in the presence of human glioma cells, but not primary human astrocytes, TIME cells are induced to form stable tubules. Detachment of TIME cells from extracellular matrix leads to a form of programmed cell death known as anoikis. Conditional activation of the protein kinase Akt (Akt:ER*) significantly inhibited the onset of TIME cell anoikis under these conditions. We believe that the ability of hTERT to immortalize primary human endothelial cells, and the fact that such cells retain the endothelial characteristics of the cells from which they were derived, will greatly facilitate the analysis of human endothelial cell biology in vitro.

Androgen induces differentiation of a human papillomavirus 16 E6/E7 immortalized prostate epithelial cell line.

Androgen signaling is crucial for the growth and development, as well as for tumorigenesis of the prostate. However, many of the prostate epithelial cell lines developed previously, either normal or tumorigenic, do not express androgen receptor (AR) or respond to androgen. In order to advance our understanding on how androgen signaling regulates the growth and the differentiation status, and affects tumorigenicity of the epithelial cell, we performed experiments on HPr-1, a prostate cell line recently immortalized from normal human prostate epithelial cells. In the present study, AR was stably transfected into HPr-1 cells by replication-defective retrovirus. Treatment of HPr-1AR cells with androgen resulted in cell differentiation and growth retardation accompanied with up-regulation of cytokeratins K8 and K18, prostate specific antigen, p21 and p27, and down-regulation of c-myc, bcl-2 and telomerase activity. Our results suggest that androgen promotes the process of differentiation in a human papillomavirus 16 E6/E7 immortalized prostate epithelial cell line which may reflect the normal effects of androgen on prostate cells.

Retargeting of viral vectors to the folate receptor endocytic pathway

Viral vectors with high transfection efficiencies are not always those with optimal target cell binding specificities. As a consequence, virus pseudotyping has been developed to endow transfection competent viruses with improved cell binding specificities and affinities. We have hypothesized that chemical conjugation of a virus to a cell specific ligand might also alter its target cell specificity and produce a virus that would transfect only the desired cell type. To test this concept, an ecotropic replication-defective myeloproliferative sarcoma retrovirus and an amphotropic murine adenovirus containing the gene for β-galactosidase were chemically derivatized with folic acid. As expected from its strong ecotropism, the unmodified retrovirus did not induce β-galactosidase expression in nonhost KB cells, while the amphotropic adenovirus yielded high levels of gene expression in the same cell line. Surprisingly, although folate derivatization enabled avid binding of both viruses to folate receptor expressing KB cells, the folate conjugation did not promote retroviral gene expression and actually prevented the normal β-galactosidase expression seen with the adenoviral vector. The fact that co-administration of excess free folic acid to block uptake by folate receptor-mediated endocytosis restored adenoviral gene expression to the level obtained with unmodified virus suggests that folate derivatization per se does not hamper viral activity. We, therefore, conclude that neither retroviral nor adenoviral delivery via the folate endocytosis pathway is compatible with viral gene expression in KB cells.

A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors.

A major obstacle in cancer gene therapy is the limited efficiency of in vivo gene transfer by replication-defective retrovirus vectors in current use. One strategy for circumventing this difficulty would be to use vectors capable of replication within tumor tissues. We have developed a replication-competent retrovirus (RCR) vector derived from murine leukemia virus (MuLV). This vector utilizes a unique design strategy in which an internal ribosome entry site-transgene cassette is positioned between the env gene and the 3' long terminal repeat (LTR). The ability of this vector to replicate and transmit a transgene was examined in culture and in a solid tumor model in vivo. The RCR vector exhibited replication kinetics similar to those of wildtype MuLV and mediated efficient delivery of the transgene throughout an entire population of cells in culture after an initial inoculation with 1 plaque-forming unit (PFU) of vector per 2000 cells. After injection of 6 x 10(3) PFU of vector into established subcutaneous tumors, highly efficient spread of the transgene was observed over a period of 7 weeks, in some cases resulting in spread of the transgene throughout the entire tumor. MuLV-based RCR vectors show significant advantages over standard replication-defective vectors in efficiency of gene delivery both in culture and in vivo. This represents the first example of the use of an RCR vector in an adult mammalian host, and their first application to transduction of solid tumors.

Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors.

The olfactory bulb, neocortex and archicortex arise from a common pool of progenitors in the dorsal telencephalon. We studied the consequences of supplying excess Notch1 signal in vivo on the cellular and regional destinies of telencephalic precursors using bicistronic replication defective retroviruses. After ventricular injections mid-neurogenesis (E14.5), activated Notch1 retrovirus markedly inhibited the generation of neurons from telencephalic precursors, delayed the emergence of cells from the subventricular zone (SVZ), and produced an augmentation of glial progeny in the neo- and archicortex. However, activated Notch1 had a distinct effect on the progenitors of the olfactory bulb, markedly reducing the numbers of cells of any type that migrated there. To elucidate the mechanism of the cell fate changes elicited by Notch1 signals in the cortical regions, short- and long-term cultures of E14.5 telencephalic progenitors were examined. These studies reveal that activated Notch1 elicits a cessation of proliferation that coincides with an inhibition of the generation of neurons. Later, during gliogenesis, activated Notch1 triggers a rapid cellular proliferation with a significant increase in the generation of cells expressing GFAP. To examine the generation of cells destined for the olfactory bulb, we used stereotaxic injections into the early postnatal anterior subventricular zone (SVZa). We observed that precursors of the olfactory bulb responded to Notch signals by remaining quiescent and failing to give rise to differentiated progeny of any type, unlike cortical precursor cells, which generated glia instead of neurons. These data show that forebrain precursors vary in their response to Notch signals according to spatial and temporal cues, and that Notch signals influence the composition of forebrain regions by modulating the rate of proliferation of neural precursor cells.

Evolution and characterization of tetraonine endogenous retrovirus: a new virus related to avian sarcoma and leukosis viruses.

In a previous study, we found avian sarcoma and leukosis virus (ASLV) gag genes in 19 species of birds in the order Galliformes including all grouse and ptarmigan (Tetraoninae) surveyed. Our data suggested that retroviruses had been transmitted horizontally among some host species. To further investigate these elements, we sequenced a replication-defective retrovirus, here named tetraonine endogenous retrovirus (TERV), from Bonasa umbellus (ruffed grouse). This is the first report of a complete, replication-defective ASLV provirus sequence from any bird other than the domestic chicken. We found a replication-defective proviral sequence consisting of putative Gag and Env proteins flanked by long terminal repeats. Reverse transcription-PCR analysis showed that retroviral gag sequences closely related to TERV are transcribed, supporting the hypothesis that TERV is an active endogenous retrovirus. Phylogenetic analyses suggest that TERV may have arisen via recombination between different retroviral lineages infecting birds. Southern blotting using gag probes showed that TERV occurs in tetraonines but not in chickens or ducks, suggesting that integration occurred after the earliest phasianid divergences but prior to the radiation of tetraonine birds.

Retroviral-mediated gene transduction.

AbstractAn obligatory part of the life cycle of retroviruses (Fig. 1) is a stable, chromosomally integrated form of the virus, known as the provirus. The existence of the proviral form of retroviruses has provided one of the main driving forces for their use as vectors for gene transfer because, in the instance of a replication-defective retrovirus vector, the proviral form is the end point of the transduction (infection) process of the target cell (Fig. 2). The use of (replication) defective retroviral vectors is therefore ideal where the goal is the stable genetic modification of the target cell. The term transduction is now used to describe the infection of a cell with such a replication-defective retrovirus. Although replication-competent retroviral vectors have also been made, these are not in general use and offer little to recommend them. In this chapter vector should be taken as referring specifically to replication-defective retroviral vectors. Retroviral life cycle. Virus particles bind to the cell via specific plasma membrane receptors resulting in endocytosis and virus disassembly. The process of conversion of the single-stranded RNA genome of the virus into double-stranded DNA (dsDNA) via reverse transcription takes place in the cytoplasm. A complex containing dsDNA and retroviral proteins then gains access to the nucleus during cell division and the dsDNA molecule integrates into the chromosome to give the proviral form. This is then transcribed to give mRNAs encoding Gag/Pol and Env proteins. After proteolytic processing, these proteins are assembled into virions along with the viral genomic RNA and virus particles released by budding. Recombinant retroviral vector transduction. The initial stages of the process, up to provirus formation, are the same as for wild-type virus (see Fig. 1 ). However, because the recombinant vector encodes no functional viral proteins, no virus can be produced. Proviral transcripts simply serve to encode (depending on the exact design of the vector) the gene of interest. KeywordsTransient ExpressionClonal Cell LineHelper VirusProducer Cell LinePackaging Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Issues in the manufacture and transplantation of genetically modified hematopoietic stem cells.

The advent of safe and practical means to correct, enhance or protect blood cells at the genetic level offers tantalizing therapeutic perspectives. At present, gene delivery using a replication-defective retrovirus is the most efficient method to stably transduce hematopoietic cells. The successful adaptation of retroviral infection to hematopoietic stem cells requires optimized transduction conditions that maximize gene transfer while preserving the cells' potential for engraftment and longterm hematopoiesis. The successful establishment of effective transduction protocols hinges on retrovirus biology as well as stem cell and transplantation biology. Interestingly, the genetic approach could permit novel strategies to promote host repopulation by transplanted stem cells. However, regulated and predictable expression of any transgene integrated at random chromosomal locations cannot be taken for granted. Investigation of the control of transgene expression and prevention of vector silencing will become increasingly important.

Retrovirus-Mediated Delivery of HPV16 E7 Antisense RNA Inhibited Tumorigenicity of CaSki Cells

Objective. In cervical cancer, high-risk human papillomavirus (HPV) genes are expressed solely in cancerous cells and have been proposed to be the most important etiological factors for cervical cancer, thus making them suitable targets for gene therapy. In this study, we aim to inactivate the HPV16 E7 in CaSki cells and test the possibility of reducing the tumorigenicity of these cells.Methods. The full-length HPV16 E7 cDNA was cloned in the pBabe-puro or pWZL-Hygro retrovirus vector in reverse orientation and was stably transfected into CaSki cells by replication-defective retrovirus infection giving rise to CaSki-E7AS and CaSki-E7AS2X cells. Immunoprecipitation/Western analysis and real-time RT-PCR were performed to document the levels of HPV16 E7 gene product. Flow cytometry was performed to study changes in the cell cycle in response to reduced E7 protein. The expression of bcl-2, RB, and E2F-1 was studied using Western blot analysis. Tumorigenicity of CaSki, CaSki-E7AS, and CaSki-E7AS2X cells was assayed with subepidermal tumor growth in nude mice.Results. We have documented that the delivery of the antisense gene construct resulted in the reduction of HPV16 E7 protein expression and cell proliferation in CaSki cells. Furthermore, we demonstrated that these changes were accompanied by cell cycle arrest, up-regulation of RB, and down-regulation of E2F-1 and bcl-2 proteins. More importantly, dose-dependent transduction of the antisense HPV16E7 construct was able to inhibit and/or retard the tumorigenicity of CaSki cells in vivo.Conclusions. Down-regulation of HPV16 E7 with antisense RNA is beneficial in reducing the tumorigenicity of CaSki cells and can potentially be useful for HPV-associated malignancy gene therapy.

Local plasminogen activator inhibitor type 1 overexpression in rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening.

Elevated levels of plasminogen activator inhibitor type 1 (PAI-1) are found in advanced atherosclerotic plaque compared with normal vessel and may contribute to plaque progression and complications associated with plaque rupture. Increased expression of PAI-1 probably contributes to the thrombotic properties of advanced atherosclerotic plaque by impeding plasmin generation and degradation of fibrin. To test this hypothesis, we have deliberately created synthetic neointimas by seeding onto the denuded luminal surface of rat carotid arteries smooth muscle cells transduced with replication-defective retrovirus encoding rat PAI-1. This cell-based gene transfer method results in stable, long-term, and localized gene expression. PAI-1 overexpression increases mural thrombus accumulation at 4 days but decreases neointimal area by 30% and 25% at 1 week and 2 weeks, respectively. PAI-1 overexpression accelerates reendothelialization of injured arteries compared with control arteries at 1 week, 2 weeks, and 1 month. PAI-1 overexpression does not alter matrix accumulation at 1 week. Increased PAI-1 expression in the rat carotid artery enhances thrombosis and endothelial regeneration while inhibiting intimal thickening. These results suggest that PAI-1 could play a direct role in the development of advanced atherosclerotic plaque and in the repair of the diseased vessel after fibrous cap disruption.

Retroviral vector-producer cell mediated angiogenesis inhibition restricts neuroblastoma growth in vivo

The purpose of this study was to determine whether gene therapy-mediated delivery of an angiogenesis inhibitor, a truncated, soluble vascular endothelial growth factor receptor (Flk-1/KDR, VEGFR-2), could suppress tumor growth in a murine model of neuroblastoma. Murine fibroblasts producing a replication-defective retrovirus encoding this mutant form of flk-1 were made. These producer cells were mixed with neuroblastoma cells and injected subcutaneously into SCID mice. Subsequent tumor growth was then measured. Murine neuroblastoma growth was decreased by 95% after 25 days. Similar tumor growth inhibitory effects were observed when the flk-1 producer cells were co-injected with cells from two different human neuroblastoma cell lines. Neuroblastoma growth can be significantly restricted in vivo with a single injection of cells that produce a retroviral vector encoding the gene for an angiogenesis inhibitor. This suggests that gene therapy-mediated delivery can be an effective alternative to chronic administration of these cytostatic anticancer agents.

Characterization of Recombination Events Leading to the Production of an Ecotropic Replication-Competent Retrovirus in a GP+envAM12-Derived Producer Cell Line

Replication-competent retrovirus (RCR) was identified in a GP+envAM12-derived producer cell, containing the MFG-S-Neo retroviral vector, using a marker rescue assay. Studies were undertaken to determine the origin and structure of this RCR. Receptor interference assays demonstrated that the virus was pseudotyped with an ecotropic envelope. Molecular analysis demonstrated the presence of a MoMLV ecotropic env recombinant where the neomycin resistance gene of the MFG-S-Neo vector was replaced by MoMLV ecotropic env. Additional recombinants linking the retroviral pol gene to neo and the neo gene to MoMLV env were also identified. A full-length MoMLV retroviral genome was detected by nested PCR in the contaminated amphotropic producer cells and in cells infected with its supernatant. Unexpectedly, this was also present in the GP+E86 packaging cells together with a previously undescribed envelope construct possessing a full 5′ and 3′ LTR, although these cells were consistently negative for the presence of RCR. These anomalies in the GP+E86 packaging cell line result in increased homology with the MFG-S-Neo vector, leading to an increased risk for the production of RCR. Our findings point to a need for increased vigilance when using these packaging lines to generate replication-defective retrovirus.

Use of recombinant viruses to assess the pattern of early human immunodeficiency virus breakthrough infection in the presence of stavudine.

A variety of cell lines were infected with replication-defective recombinant retroviruses in the presence of stavudine (d4T). Cells which were infected despite the presence of d4T were isolated and subjected to infection with other retroviruses [replication-competent human immunodeficiency virus (HIV), replication-defective HIV or replication-defective recombinant murine retroviruses]. Each of the host cell types tested had a small subset of cells that were infected with HIV or murine retroviruses in the presence of d4T. Some of these infected cells could be infected repeatedly at high efficiency in the presence of d4T. This phenotype of 'persistent refractoriness' to the antiviral effects of d4T could be overcome by the addition of 5-fluoro-2-deoxyuridine (floxuridine) to d4T. The d4T-floxuridine combination also had potent antiretroviral effects in primary blood mononuclear cells.

Bone marrow stromal cells as a vehicle for gene transfer.

Adoptive transfer of genetically modified somatic cells is playing an increasingly important role in the management of a wide spectrum of human diseases. Hematopoietic stem cells and lymphocytes have been used to transfer a variety of genes, however, they have limitations. In this study, the feasibility of retroviral gene transduction of bone marrow stromal cells, and the engraftment characteristics of these cells following infusion, was investigated in a murine transplantation model. Stromal cells derived from Balb/c mouse bone marrow were transduced with a replication-defective retrovirus containing the LacZ gene. Following three rounds of transduction, between 5 and 40% of the cells were positive for the LacZ gene. A total of 2 x 106 cells were infused into the same mouse strain. After the infusion, the LacZ gene was detected by PCR in the bone marrow, spleen, liver, kidney and lung; however, only the spleen and bone marrow samples were strongly positive. Quantitative PCR demonstrated that between 3 and 5% of spleen and bone marrow cells, and 1% of liver cells contained the LacZ gene at 3 weeks after infusion; <0.2% transduced cells were found in other organs. No difference was noted in engraftment between mice with or without irradiation before transplantation, suggesting that engraftment occurred without myeloablation. The infused transduced cells persisted for up to 24 weeks. Self-renewal of transplanted stromal cells was demonstrated in secondary transplant studies. Ease of culture and gene transduction and tissue specificity to hematopoietic organs (bone marrow, spleen, liver) is demonstrated, indicating that stromal cells may be an ideal vehicle for gene transfer.

Immobilized Sample Amplification for Quantitative Determination of Retroviruses

Immobilized sample amplification (ISA) is a novel method for amplification, detection, monitoring, and quantitative determination of nucleic acids from a minute amount of sample. We present here a novel quantitative ISA assay for retroviruses using a replication-defective recombinant retrovirus as a model retrovirus. Samples, as small as 5 to 10 μl or as large as 1 ml or more in volume, are readily immobilized on a nylon or polyester matrix. Retroviral RNA is directly amplified following the rehydration of the immobilized samples, thus eliminating the needs for retroviral RNA extraction. An ISA assay of a 10-μl viral sample generates results equal to or better than that of RT–PCR on equivalent amount RNA isolated from larger sample volumes. Recovery of RNA from small volumes, such as 10 μl, is almost impossible, whereas ISA assay detects retroviruses from as small as 1 to 5 μl of viral samples containing 104cfu/ml determined by colony-forming assay. Extraction of RNA from a small amount of infectious viral samples not only is a difficult, biohazardous procedure, but also introduces random errors which contribute to variability in viral quantitation. Since the ISA method eliminates the isolation/extraction of the nucleic acids, it significantly shortens the handling time for the biohazardous materials and simplifies the procedure for analyzing small quantities of biological samples. This method detects less than 10 infectious retroviral particles as determined by both colony-forming assay and electron microscope studies. The format and protocol of this quantitative ISA assay can be easily automated to fit into numerous platforms, thus making it attractive for laboratory automation.

Unstable retrovirus mutants with acquired transforming activity: rapid changes in the number of repeats of a specific junD polynucleotide segment.

We have previously reported that the non-transforming jun D (wild type) gene can acquire transforming activity through spontaneous mutations when it is replicated through avian replication-competent retrovirus vectors in chicken embryo fibroblasts. In two of these spontaneous mutants, T1 and T2, which were isolated from proviral DNA in the same transformed cell clone, a specific 48 bp polynucleotide segment of the jun D coding sequence was tandemly repeated three and five times, respectively. We report here that the number of direct repeats in these mutants rapidly changes (mostly decreases) in the context of either RSV-based replication-competent or MLV-based replication-defective retroviruses, most likely during the process of reverse transcription, while these mutations are stable in the cellular chromosome. We also show that the growth conditions of the infected culture modulate the proportions of polymorphic proviral populations in the infected culture. We finally discuss the possible molecular mechanisms that generate genetic diversity in these amplification mutants.

Overexpression of Protein Kinase C α and β1 Has Distinct Effects on Bovine Aortic Endothelial Cell Growth

Protein kinase C (PKC) plays an important role in the mitogenic response of endothelial cells to growth factors. PKC α and β1 are the predominant classical isoforms expressed by bovine aortic endothelial cells (BAECs). The present studies were undertaken to elucidate the effect of PKC α and β1 overexpression in BAEC growth. A series of BAEC lines that stably overexpress the full-length PKC α and β1 cDNA were generated by using a replication-defective recombinant retrovirus. The level of PKC α and β1 cDNA expression was determined by assaying for PKC α and β1 mRNA transcripts. PKC α and β1 protein levels were analysed by Western blotting. Functional analysis of these overexpressing lines was performed by measuring PKC activity and phorbol ester-binding assays. PKC α and β1 overexpression had distinctive effects on BAEC growth and cell-cycle progression. Relative to untransfected BAECs and BAECs transfected with the viral vector alone, BAECs that overproduced PKC α exhibited reduced proliferation in vitro and increased accumulation of cells in the G 2/M phase of the cell cycle. Growth inhibition was greater in cell lines overexpressing higher levels of PKC α. Conversely, a 5-fold greater increase in PKC β1 activity promoted BAEC growth and shortened BAEC doubling time, whereas cells with a 2- to 4-fold increase in enzyme activity had growth profiles similar to those of both control groups. These results suggest that PKC α and β1 overexpression has reciprocal effects on BAEC growth.

High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous retinoid X receptor.

Our studies of the Bombyx mori ecdysone receptor (BE) revealed that, unlike the Drosophila melanogaster ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulated high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with both the ultraspiracle gene product (Usp) and retinoid X receptor heterodimer partners indicated that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. We have mapped this "gain of function" to determinants within the D and E domains of BE and demonstrated that, although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulated separate "reporter" viruses in all cell types examined, suggesting that BE has potentially broad utility in the modulation of transgene expression in mammalian cells.